T1r hetero-oligomeric taste receptors, cell lines that express said receptors and taste compounds

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T1R HETERO-OLIGOMERIC TASTE RECEPTOR BINDING COMPOUNDS AND METHODS OF USE THEREOF ,ΓΡΊηιχήηΝ-ηυΓ) T1R tw mnwpon τιπίοίπ inn wwwb row 173,552/2

It is to be noted that only the subject matter embraced in the scope of the claims appended hereto, whether in the manner defined in the claims or in a manner similar thereto and involving the main features as defined in the claims, is intended to be included in the scope of the present invention, while subject matter described and exemplified to provide background and better understanding of the invention, is not intended for inclusions as part of the present invention.

The present invention in part relates to the discovery that the T1R receptors assemble to form functional taste receptors. Particularly, it has been discovered that coexpression of T1R1 and T1R3 results in a taste receptor that responds to umami taste stimuli, including monosodium glutamate. Also, it has been discovered (hat coexpression of the T1R2 and T1R3 receptors results in a taste receptor that responds to sweet taste stimuli including naturally occurring the artificial sweeteners.

Also, the present invention relates to the use of a non-naturally occurring compound that specifically binds to a hetero-oligomeric taste receptor composed of hTIRJ/hTlR3 subunits having a potency as umami flavour taste tastant, enhancer or modifier as determined by an EC50 of less than 1 mM.

The invention also relates to chimeras and truncated versions of T1R1, T1R2, and T1R3, as well as chimeras of T1R1/T1R3 and T1R2/T1R3 receptors comprising human, rat, or human and rat subunits.

Further, the invention relates to the constructions of cell lines that stably or transiently co-express a combination of T1R1 and T1R3; or T1R2 and T1R3, including truncated or chimeric versions of these subunits as well as chimeric receptors comprising wild-type or chimeric subunits; under constitutive or inducible conditions.

The use of these cell lines in cell-based assays to identify umami and sweet taste modulatory compounds is also provided, particularly high throughput screening assays that detect receptor activity by the use of fluorometric imaging.

The invention also relates to compounds that bind to T1R1/T1R3, T1R2/T1R3 receptors, as well as T1R1, T1R2, and T1R3 chimeric and truncated subunits and chimeric receptors. 1

Description of the Related Art

The taste system provides sensory information about the chemical composition of the external world. Mammals are believed to have at least five basic taste modalities: sweet, bitter, sour, salty, and umami. See, e.g., Kawamura et ai, introduction to Umami: A Basic Taste (1987); Kinnamon et αΙ.,Αηη. Rev. Physiol., 54:715-31 (1992); Lindemann, Physiol. Rev., 76:718-66 (1996); Stewart et al., Am. J. Physiol., 272:1-26(1997). Each taste modality is thought to be mediated by a distinct protein receptor or receptors that are expressed in taste receptor cells found on the surface ofthe tongue (Lindemann, Phy sol. Rev. 76:718-716 (1996)). The taste receptors that recognize bitter, sweet, and umami taste stimuli belong to the G-protein-coupled receptor (GPCR) superfamily (Hoon et al., Cell 96:451 (1999); Adler et al., Cell 100:693 (2000)). (Other taste modalities are believed to be mediated by ion channels.) G protein-coupled receptors mediate many other physiological functions, such as endocrine function, exocrine function, heart rate, lipolysis, and carbohydrate metabolism. The biochemical analysis and molecular cloning of a number of such receptors has revealed many basic principles regarding the function of these receptors. For example, United States Patent No. 5,691,188 describes how upon a ligand binding to a GPCR, the receptor undergoes a conformational change leading to activation of a heterotrimeric G protein by promoting the displacement of bound GDP by GTP on the surface of the Ga subunit and subsequent dissociation of the Ga subunit from the Gp and Gy subunits. The free Ga subunits and Gpy complexes activate downstream elements of a variety of signal transduction pathways.

The T1R receptors were previously hypothesized to function as sweet taste receptors (Hoon et al., Cell 96:541-51 (1999); Kitagawa et al., Biochem Biophys Res. Commun. 283:236-42 (2001); Max et al., Nat. Genet. 28:58-63 (2001); Montmayeur et al., Nat. Neurosci. 4: 412-8 (2001); Sainz et al., J. Neurochem. 77: 896-903 (2001)), and Nelson et al. (2001) and Li et al (2002) have recently demonstrated that rat and human, respectively, T1R2 and T1R3 act in combination to recognize sweet taste stimuli.

However, there remains in the art a need for new and improved flavoring agents. For example, one of the five known basic tastes is the “savory” or “umami” 2 173,552/2 flavor of monosodium glutamate (“MSG”). MSG is known to produce adverse reactions in some people, but very little progress has been made in identifying artificial substitutes for MSG. It is known that a few naturally occurring materials can increase or enhance the effectiveness of MSG as a savory flavoring agent, so that less MSG would be needed for a given flavoring application. For example the naturally occurring nucleotide compounds inosine monophosphate (IMP) or guanosine monophosphate (GMP) are known to have multiplier effect on the savory taste of MSG, but IMP and GMO are very difficult and expensive to isolate and purify from natural sources, or synthesize, and hence have only limited practical application to most commercial needs in food or medical compositions. Less expensive compounds that would provide the flavor of MSG itself, or enhance the effectiveness of any MSG that is present could be of very high value. Similarly, discovery of compounds that are either new “High Intensity” sweeteners (i.e. they are many times sweeter than sucrose) would be of value.

What is needed in the art is the identification and characterization of taste receptors which function as sweet and umami receptors, assays for identifying compounds that modulate (enhance or block) sweet and umami taste, and the compounds that specifically bind to these receptors.

Summary of the Invention

The present invention provides a chimeric receptor comprising various combinations of human and rat TIRs, such as a chimeric TIR2/TIR3 receptor comprising a human TIR2 subunit and a rat TIR3 subunit; a chimeric TIR2/TIR3 receptor comprising a rat TIR2 subunit an a human TIR3 subunit; a chimeric TIR2 receptor subunit comprising a human extracellular domain, a rat transmembrane domain and a rat intracellular domain; and a chimeric TIR3 receptor subunit comprising a rat extracellular domain, a human transmembrane domain and a human intracellular domain.

The present invention also provides a comestible or medicinal product comprising a compound as herein described.

The present invention also provides a method for modulating the savory taste of a comestible or medicinal product comprising: providing at least one comestible or medicinal product, or a precursor thereof, and combining the comestible or medicinal product or precursor thereof with at least a savory flavor modulating amount of at least one non-naturally occurring compound as herein described, or a comestibly acceptable 3 173,552/2 salt thereof, so as to form a modified comestible or medicinal product; thereby modulating the savory taste of a comestible or medicinal product.

The present invention provides a non-naturally occurring oxalamide compound that specifically binds to a chimeric hetero-oligomeric taste receptor composed of hTlR2/hTlR3 subunits having a potency as a sweet agonist as determined by an EC50 of less thanlO μΜ.

The present invention also provides a method for modulating the sweet taste of a comestible or medicinal product comprising: providing at least one comestible or medicinal product, or a precursor thereof, and combining the comestible or medicinal product or precursor thereof with at least a sweet flavor modulating amount of at least one non-naturally occurring oxalamide compound as herein described, or a comestibly acceptable salt thereof, so as to form a modified comestible or medicinal product; thereby modulating the sweet taste of a comestible or medicinal product.

Brief Description of the Figures

Figure 1 contains a sequence alignment of human and rat TIRs, human calcium-sensing receptor and rat metabotropic glutamate receptor.

Figure 2 contains RT-PCR amplification experimental results which show that hTIR2 and hTIR3 are expressed in taste tissue.

Figure 3a - 3b contain functional data (intracellular calcium responses) elicited by different sweet taste stimuli in HEK cells stably expressing Gais that are transiently transfected with human TIR2, TIR3 and TIR2/TIR3 at various concentrations of sweet taste stimuli (Figure 3a); human TIR2/TIR3 dose responses for several sweet taste stimuli (Figure 3b); human TIR2/TIR3 responses to sucrose to 4 the presence of gurmarin, and endogenous p2-adrenergic receptor responses to isoproterenol in the presence of gurmarin. Figure 3c contains the normalized response to different sweeteners.

Figure 4 contains intracellular calcium responses in HEK cells stably expressing Gal5, transiently transfected with hTlR2/hTlR3, rT!R2/rTlR3, hTlR2/rTlR3 and rTlR2/hTlR3 in response to 350 mM sucrose, 25 mM tryptophan, 15 mM aspartame, and 0.05 % monellin.

Figure 5 contains the results of a fluorescence plate reactor based assay wherein HEK cells stably expressing Gal 5 were transiently transfected with hTlR2 and hTlR3 or hTlR3 alone and contacted with the calcium dye Fluo-4 and a sweet taste stimulus (12.5 mM cyclamate).

Figure 6 contains normalized dose-response curves which show that hTlR2 and hTlR3 function in combination as the human sweet receptor based on their dose-specific interaction with various sweet stimuli (trp, cyclamate, sucrose, neotame, asparame, saccharin and Acek).

Figure 7 contains structural information relating to mGluRl and T1R1 showing the key, ligand binding residues are observed in these molecules.

Figure 8a-8c contains functional data showing HEK cells which stably express Gal 5 that are transiently transfected with T1R1/T1R3 respond to glutamate in an intracellular calcium-based assay. Figure 8a shows that intracellular calcium increases in response to increasing glutamate concentration; Figure 8b shows intracellular calcium responds to IMP (2 mM), glutamate (0.5 mM) and 0.2 mM IMP; and Figure 8c shows human T1R1/T1R3 responses for glutamate in the presence and absence of 0.2 mM IMP.

Figures 9a-9b respectively contain the results of an immunofluorescence staining assay using Myc-tagged hTlR2 and a FACS experiment showing that the incorporation of the PDZIP peptide (SEQ ID No: 1) enhanced the expression of a T1R (hTlR2) on the plasma membrane.

Figure 10a through 10b contain calcium imaging data demonstrating that hlTR2/hTlR3 respond to different sweet stimuli.

Figure 11 shows the responses of cell lines which stably express hTlRl/hTlR3 by automated fluorescence imaging to umami taste stimuli. 5

Figure 12 shows the responses of a cell line which stably expresses hTlR2/hTlR3 by automated fluorescence imaging to sweet taste stimuli.

Figure 13 shows dose-response curves determined using automated fluorescence imaging for a cell line that inducibly expresses the human T1R1/T1R3 taste receptor for L-glutamate in the presence and absence of 0.2mM IMP.

Figures 14 and 15 show the response of a cell line that inducibly expresses the human T1R1/T1R3 taste receptor (1-17 clone) to a panel of L-amino acids. In Figure 14 different C-amino acids at lOmM were tested in the presence and absence of 1 mM IMP. In Figure 15 dose-responses for active amino acids were determined in the presence of 0.2mM IMP.

Figure 16 shows that lactisole inhibits the receptor activities of human T1R2/T1R3 and human T1R1/T1R3.

Figure 17 shows schematics of human-rat T1R chimeras. The chimeras are constructed by fusing the human or rat extracellular domains to the rat or human transmembrane domains respectively, as shown in h2-r2, r2-h2, h3-r3 and r3-h3.

Figure 18 shows neohesperidin dihydrochalcone (NHDC) enhances the activities of T1R1/T1R3 umami taste receptor. [Neohesperidin dihydrochalcone] = 5 μΜ. The glutamate dose response curve is left-shifted by 2.3 fold (left panel), and the glutamate/IMP dose response is left-shifted by 2.1 fold.

Figure 19 shows that control sweeteners do not affect the activities of T1R1/T1R3 umami taste receptor [Steviocide] = 0.5 mM. [Saccharin] = 1 mM. Glutamate dose response is shown in the left panel, and glutamate/IMP dose response is shown in the right panel.

Figure 20 shows NHDC maps to the transmembrane domain of human T1R3.

Figure 21 shows mapping of a compound to the human T1R2 transmembrane domain.

Figures 22a-d show sweeteners which map to different domains/subunits of the human sweet receptor. Figure 22a shows responses of human and rat sweet receptors to sucrose (200 mM), aspartame (10 mM), neotame (0.1 mM), cyclamate (10 mM), and sucrose (200 mM) in the presence of lactisole (1 mM) (Suc/Lac). HEK-293T cells were transiently transfected with human or rat T1R2, T1R3, and a G«i5 chimera Gais/u, and assayed for intracellular calcium increases in response to sweeteners. Figure 22b shows aspartame and neotame were mapped to N- terminal extracellular domain of human 6 T1R2, Combinations of T1R chimeras were transiently transfected into HEK-293T cells with Gais/n, and assayed for responses to sweeteners at the concentrations listed in 23a. The presence or absence of response is what is important. Figure 22c shows cyclamate was mapped to the C-terminal transmembrane domain of human T1R3. Figure 22d shows lactisole was mapped to the transmembrane domain of human T1R3. Different combinations of T1R chimeras were transiently transfected into HEK-293T cells with G Gaii/ii, and assayed for responses to sucrose (200 mM) and AceK (10 mM) in the absence or presence of lactisole (1 mM). The activities in B, C and D represent the mean ± SE of number of responding cells for four imaged field of-1,000 confluent cells.

Figures 23a-d show mutations in T1R2 or T1R3 selectively affect the activity of different sweeteners. Figure 23a shows sequence alignment of the N-terminal ligand binding domain of rat mGluR5 with human and rodent TlR2s. The 8 critical amino acids involved in ligand-binding in mGluR5 are labeled with *, three of the 8 amino acids are conserved in T1R2 and underlined. Figure 23b shows two point mutations in the human T1R2 N-terminal extracellular domain that abolish response to aspartame and neotame without affecting cyclamate. Stable cell lines of hTlR2/hTlR3 (WT), hTlR2 S144A/hTlR3 (S144A) and hTlR2 E302A/hTlR3 (E302A) were generated as describe in the Examples. The dose-responses of these stable lines were determined on FLIPR for sucrose, aspartame, neotame and cyclamate. The activities represent the mean ± SE of fold increases in fluorescence intensities for four recorded wells. Figure 23c shows sequence alignment of human and rodent T1R3 transmembrane domains. The three extracellular loops are underlined and labeled ELI, 2, or 3, according to their order in the protein sequences. Figure 23d shows mutations in the extracellular loop of hTlR3 that abolish response to cyclamate without affecting aspartame. Each of the three extracellular loops of hTlR3 were replaced with rat protein sequence separately, and the resulting hTlR3 mutants were transiently transfected into HEK-293T cells together with G uis/n, and assayed for responses to sucrose (200mM), aspartame (10 mM) and cyclamate (10 mM). The activities represent the mean ± SE of number of responding cells for four imaged field of -1,000 confluent cells.

Figures 24a-b show human T1R2 is required for Gais -coupling. Figure 24a shows responses of human, rat and chimeric sweet receptors to sucrose (200 mM) and AceK (10 mM). Stable Gais cells were transiently transfected with human, rat or 7 chimeric TIRs, and assayed for intracellular calcium increases in response to sweeteners. Figure 24b shows Gais-coupling is mediated by human T1R2. The activities represent the mean ± SE of number of responding cells for four imaged field of -1,000 confluent cells.

Figures 25a-f show the effect of lactisole and cyclamate on the human T1R1/T1R3 umami receptor. Figure 25a shows the response of human T1R1/T1R3 stable cell line to L-glutamate (5 mM) and L- glutamate/IMP (1/0.2 mM) in the absence and presence of lactisole (5 mM). Figure 25b shows the lactisole dose-dependent inhibition curves were determined for L-glutamate (Glu), and L- glutamate with 0.2 mM IMP (Glu/IMP), each at two different concentrations. The IC50s are 0.19 ± 0.02 mM and 0.21 ± 0.01 mM for L-glutamate at 8 and 80 mM; 0.35 ± 0.03 mM and 0.82 ± 0.06 mM for L-glutamate with IMP at 0,8 and 8 mM respectively. Figure 25c shows the dose responses for L-glutamate, with or without 0.2 mM IMP, were determined in the presence of different concentrations of lactisole. In the presence of 0, 25, or 50 μΜ lactisole, the EC50s are 9.9 ± 1.5 mM, 7.9 + 0.5 mM, and 7.0 ± 0.3 mM for L-glutamate; in the presence of 0, 100, or 200 μΜ lactisole, the ECsos are 0.53 ± 0.04 mM, 0.71 ± 0.10 mM, and 0.84 ± 0.10 mM for L-glutamate with IMP. Values represent the mean ± SE for four independent responses. Figure 25d shows the detection thresholds for sweet, umami, and salty taste stimuli were determined in the presence or absence of lactisole. The inhibition effect of lactisole is shown as fold increases in detection thresholds, “Detection thresholds” are defined as the lower limit of detectable tastants. The detection threshold values were averaged over four trials for three subjects. Figure 25e shows the responses of human T1R1/T1R3 stable cell line to threshold level of L-glutamate (4 mM) and endogenous M2 receptor agonist carbachol were assayed on FLIPR in the absence and presence of various concentrations of cyclamate. Figure 25f shows the dose-responses of the human T1R1/T1R3 stable cell line were determined on FLIPR for L-glutamate with or without 0.2 mM IMP in the absence and presence of cyclamate (8 mM). The activities in B, C, E and F represent the mean ± SE of fold increases in fluorescence intensities for four recorded wells. The dose- responses in B, C, E and F were reproduced at least 6 times independently.

Figure 26 shows a working model for the sweet and umami taste receptor structure-function relationships. Filled arrows indicate direct activation, open arrows indicate enhancement, and bar heads indicate inhibition. 8

Figure 27a shows all 16 combinations of TIRs and chimeras that were tested for responses to sweeteners and lactisole. rTlR2/TlR3H-R, rTlR2/hTlR3, and T1R2H- R/T1R3R-H show a significant response to cyclamate and they can be inhibited by lactisole. T1R chimeras were transiently transfected into HEK-293T cells with Gai5/ii. 5 The activities represent the mean ± SE of number of responding cells for four imaged field of -1,000 confluent cells, each unit on the Y axis represents 50 responding cells. Abbreviations: Sue (sucrose lOOmM); Suc/Lac (sucrose lOOmM, lactisole 1 mM);

AceK (acesulfame K 10 mM); AceK/Lac (acesulfame K 10 mM, lactisole 1 mM); ATM (aspartame lOmM); NTM (neotame 10 mM); Cyc (cyclamate 10 mM). Figure 10 27b shows the lactisole dose-dependent inhibition curves of the human sweet receptor were determined for sucrose (Sue), saccharin (Sac), and D-tryptophan (D-Trp), each at two different concentrations. The lC50s are 19.6 ± 0.1 μΜ and 64.6 ± 0.3 μΜ for sucrose at 50 mM and 120 mM; 22.6 ± 0.1 μΜ and 103 i 7 μΜ for saccharin at 0.1 and 2 mM; 19.9 ± 0.2 μΜ and 168 ± 9 μΜ for D-tryptophan respectively. Figure 27c shows 15 the dose responses of human sweet receptor for sucrose, D-Trp and saccharin were determined with different concentrations of lactisole. In the presence of 0, 10, or 20 μΜ lactisole, theECsos are 19.4 ± 0.9 mM, 24.7 ± 1.0 mM, and 31.3 ± 0.3 mM for sucrose; 0.37 ± 0.02 mM, 0.60 ± 0.03 mM, 0.94 ± 0.08 mM for D-Trp; 42 ± 3 μΜ, 67 ± 6 μΜ, 118 ± 2 μΜ for saccharin. Values represent the mean ± SE for four independent 20 responses. The dose-responses in B and C were determined at least 6 times independently, and generated similar results as shown here.

Detailed Description of the Invention

The inventions provides compounds that specifically bind to the wild-type and 25 chimeric sweet and umami taste receptors disclosed herein. Further provided are compounds that specifically bind to the wild-type, chimeric or truncated T1R2 or T1R3 subunits of the sweet and umami receptors.

Binding to the T1R2ZT1R3 sweet receptor defines a large genus of molecules. The receptor responds to every sweetener tested, including carbohydrate sugars, amino 30 acids and derivatives, sweet proteins, and synthetic sweeteners. In the meantime, the receptor exhibits stereo-selectivity for certain sweeteners, for example, it responds to D-tryptophan but not L-tryptophan, which is in correlation with taste physiology data. 9 173,552/2

Thus, the compounds of the invention specifically bind chimeric receptors. Examples include, but are not limited to, a chimeric TIR2/TIR3 receptor comprising a human TIR2 subunit and a rat TIR3 subunit; a chimeric TIR2/TIR3 receptor comprising a rat TIR2 subunit and a human TIR3 subunit; a chimeric T1R2 receptor subunit comprising a human extracellular domain, a rat transmembrane domain and a rat intracellular domain; and a chimeric TIR3 receptor subunit comprising a rat extracellular domain, a human transmembrane domain and a human intracellular domain. The invention provides functional taste receptors, preferably human taste receptors, that are produced by co-expression of a combination of different TIRs, preferably TIR1ITIR3 or TIR2/TIR3, and the corresponding isolated nucleic acid sequences or fragments, chimeras, or variants thereof that upon coexpression result in a functional taste receptor, i.e., a sweet taste receptor (TIR2/T1R3) or umami taste receptor (TIR1ITIR3). TIRs, a family of class C G protein-coupled receptors (GPCRs), are selectively expressed in the taste tissue (Roon, M.A., et al., Cell, 1999.96(4): p. 541-51, Bachmanov, A.A., et al., Chem Senses, 2001. 26(7): p. 925-33, Montmayeur, J.P, et al., Nat Neurosci, 2001. 4(5): p. 492-8, Max, M., et al., Nat Genet, 2001. 28(1): p. 58-63, Kitagawa, M., el al., Biochem Biophys Res Commun, 2001. 283(1): p. 236-42 and Nelson, G., et al, Cell, 2001. 106(3): p. 381-90.) Functional expression of TIRs in HEK293 cells revealed that different combinations of TIRs respond to sweet and umami taste stimuli (Nelson, G., et al., Cell, 2001. 106(3): p. 381-90, Li, x, et al., Proc Natl Acad Sci USA, 2002.99(7): p. 4692-6.) TIR2 and TIR3, when co-expressed in 293 cells, recognize diverse natural and synthetic sweeteners For the reason mentioned above re "diverse", please consider whether we need this section for enablement. If not, delete. We can discuss, while TIRI and TIR3 recognize umami taste stimulus L-glutamate, and this response is enhanced by 5' -ribonucleotides, a hallmark of umami taste. Knockout data confirmed that TIRs indeed mediate mouse sweet and umami tastes (Damak, S., et al. Science, 2003 301(5634): p. 850-3, Zhao, G.Q, et al. Cell 2003 Oct 31; 115(3):255-66).

The class C GPCRs possess a large N-terrninal extracellular domain, often referred to as the Venus flytrap domain (VFD) (Pin, J.P, Pharmacol Ther, 2003 98(3): p. 325-54), and are known to function as either homodimers, in the cases of metabotropic glutamate receptors (mGluRs) and calcium- sensing receptor (CaR), or 10 heterodimers, in the case of γ-aminobutyric acid type B receptor (GAB AbR). The functional expression data shows a heterodimer mechanism for TIRs: both T1R1 and T1R2 need to be coexpressed with T1R3 to be functional, which is supported by the overlapping expression patterns of TIRs in rodent tongue.

It is established herein that T1R family members act in combination with other T1R family members to function as sweet and umami taste receptors. As disclosed in further detail infra in the experimental examples, it has been demonstrated that heterologous cells which co-express hTlR2 and hTlR3 are selectively activated by sweet taste stimuli in a manner that mirrors human sweet taste.

For example, HEK-293-Gal5 cells that co-express hT!R2 and hTlR3 specifically respond to cyclamate, sucrose, aspartame, and saccharin, and the dose responses for these compounds correlate with the psychophysical taste detection thresholds.

Also, as supported by data in the experimental examples, it has been shown that cells which co-express hTIRl and hTlR3 are selectively activated by glutamate (monosodium glutamate) and 5’-ribonucleotides in a manner that mirrors human umami taste. For example, HEK-293-Gal5 cells that co-express hTIRl and hTlR3 specifically respond to glutamate and the dose response for this umami-tasting compound correlates with its psychophysical taste detection threshold. Moreover, 5’-ribonucleotides such as IMP enhance the glutamate response of the T1R1/T1R3 receptor, a synergism characteristic of umami taste.

Further, as shown by experimental data in the examples it has been shown that cells which stably and inducibly co-express T1R1/T1R3 selectively respond to the umami taste stimuli L-glutamate and L-aspartate and only weakly respond to other L-amino acids, and at much higher concentrations, providing further evidence that the T1R1/T1R3 receptor can be used in assays to identify compounds that modulate (enhance or block) umami taste stimuli.

Examples of compounds that specifically bind to the sweet receptor and modulate sweet taste can be found in Table 5.

Tables 1-4 provide examples of compounds that specifically bind to the umami receptor and modulate umami taste. 11

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Tabic 1 - Umami Amides Compound No. Compound Umami EC50(uM) Ecso ratio (vs. MSG) @ (uM) s Al 3,6-DicHoro-N-(4-ethoxy-phenyl)-2- methoxy-benzamide 0.22 2.74 1 &gt; 8 X B V A2 xc- 4-(3,6-Dichloro-2-methoxy-benzoylamino)-benzoic acid methyl ester 0.93 6.98 0.01 °\ /Λ Kt-VX 0 A3 c \=λΆ 1.08 6.14 0.03 a2,5-dichloro-N-(4- ethoxyphenyl)benzamide A4 HI IH” 2- [(Benzo [b] thiophene-2 -cai amino] -4-methyl-pentanoi methyl ester 1 -.1IIIIIH Ύ rbonyl)-c acid 0.4 J A5 HI in·' 2 - [(Benzofuran-2 -carbonyl)-a methyl-penlanoic acid meth .....IIIH 7 mino]-4-yl ester 0.31 A 0 HN· -^H A6 0.32 2.86 1 2 -[(5 -Methoxy-benzofur an-2-carbonyl)-amino] -4-methyl-pentanoic acid methyl ester 12

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Table 1 - Cm ami Amides Compound No. Compound Umami ECSo(uM) Ecjo ratio (vs. MSG) @ (uM) A19 /\ 5-mel yi) &lt;YvrO /-A '-° Xo thoxy-N-( 1 -methoxypentan-2 -benzofuran-2 -carboxamide 1.3 A20 5-methyl-N-(2-methylheptan-4-yl) benzofiiran-2 -carboxamide 1.32 A21 CM L N-(pentan-3 -yI)benzofuran-2 -carboxamide 1.52 3.74 1 A22 N·^ c Benz H 0 \ othiazole-6-carboxylic a propyl-butyl)-amide cid (1- 1.58 A23 /°ΥΊ νΜγ\ 0 2 -methyl -N-(2 -methylhepti yl)benzo[d]oxazole-5-carbox in-4- amide 0.38 A24 2-methyl-N-(2-methyIheptan-4- yl)benzo[d]oxazole-6-carboxamide 1.12 15

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Table 1 - Umami Amides Compound No. Compound Umami ECS0(uM) Ecso ratio (vs. MSG) @ (uM) A25 "oY7 (R)-4 -Methyl -2 - [(2-methyl -benzooxazole-6-carbonyl)-amino] -pentanoic acid methyl ester 1.48 A26 Y 2-methyl-N-(2-methylhexan-3 -yl)benzo [d] oxazole-6-carboxamide 1.6 A27 aY: 2-ethyl -N-(heptan-4 -yl)benzo [ d] oxazole-6-carboxamide 1.61 A28 aXYh o o (R)- 4-Methyl-2-[(2-methyl -benzooxazole-5-carbonyl)-a mino] -pentanoic acid methyl ester 1.69 A29 cbY N-(heptan-4-yl)benzo[d] oxazole-6 -carboxamide 1.91 A30 Brx _/ V X 5-bromo-N-(heptan-4-yl)furan-2- carboxainide 0.49 12.6 1 16

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Table 1 - Umami Amides Compound No. Compound Umami ECso (uM) Ecso ratio (vs. MSG) @ (uM) A36 Η Λ °v Vv^A/k 0 \ (R)-methyl 2-(lH-indole-5-carboxamido)-4-methylpentanoate 1.01 A37 \\ T f "'H ° k (R)-methyl 4-methyl-2-(quinoline-6-carboxamido)pentanoate 1.5 A38 Av 5 -Methyl-thiophene-2 -carboxylic acid (1 -propyl-butyl)-amide 1.22 6,54 1 A39 o 5-Methyl-thiophene-2-carboxylic acid (1,2,3,4 -telrahydro-naphthalen-1 -yl)-amide 1.31 2.3 1 A40 j II Η λ ·— · o (R)-methyl 2-(2-naphthamido)-4-metliylpentanoate 0.37 18

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Table 1 - Umami Amides Compound No. Compound Umami EC5o (uM) Ee® ratio (vs. MSG) I® A A A41 \A kJ H Y"1 0 0.7 2.14 3 N-(nonan-5-yI)benzo[d] [ 1,3]dioxole-5-carboxamide A Y \7 V J H / 0 A,/ 7""«Vh A42 0 0.35 (2R,3R)-methyl 2- (benzo[d][l,3]dioxole-5-cafboxamido)-3 -metliylpentanoate Λ A 7 / &lt;, J VY A43 0 0.49 2-[(Benzo[l,3]dioxole-5-carbonyl)-amino]-hexanoic acid methyl ester A 3 Ί] V/ 0-^ J J Y H / v Y A44 o &gt; 0.61 (R)-2-[(Benzo[l,3]dioxole-5-carbonyl)-amino]-hexanoic acid methyl ester \ / A45 ί ** A J k 0 Ά f 4 y-0 0.88 (R)-ethyl 2-(benzo[d3[l,3]dioxole-5-carboxamido)-4-methyIpentanoate 19

<img img-format="tif" img-content="drawing" file="IL173552AD000212.tif" id="idf0012" />

Table 1 - Umami Amides Compound No. Compound Umami EC50(uM) Ecm ratio (vs. MSG) @ (uM) A46 0 (R)-mel 5-carb V/ o \ .hyl 2-(2,3-dihydrobenzofuran-D.xamido)-4-methvlpentanoate 1.32 A47 3 (S)-N-(l ,2,3,4-letrahydronaphthalen-1 -yl)benzo[d][l,3]dioxole-5-carboxainide 1.33 6.42 0.1 A48 coY,“o 0 ' N-(4-pheny lbutan-2 -yl)benzo [d] [l,3]dioxole-5-carboxamide 1.51 9.27 1 A49 0 2-[(Benzo[l,3]dioxole-5-carbonyl)-amino]-pentanoic acid methyl ester 1.54 9.53 1 A50 c N-(t ΆΑ jenzold] [1,3]dioxol-5-yl)-2-propylpentanamide 1.57 20

<img img-format="tif" img-content="drawing" file="IL173552AD000213.tif" id="idf0013" />

Table 1 - Cm ami Amides Compound No. Compound Cmami EC50 (uM) Ecso ratio (vs. MSG) @ (uM) o r-/ V f -0 A51 7 "t, fl / H Ο ξ 1.58 (R)-propyl 2-(benzo[d][T.3]dioxole-5-carboxamido)-4 -methylpentanoate A52 H 1.65 0 N-(heptan-4-yl)-2,3-dihydrobenzofuran-5-carboxamide A53 0 H n 1.83 0 X N-(hexan-3 -y l)benzo [d J [ 1,3 ] dioxole-5-carboxamide \ fi HN— A54 \ s— / H 0.12 N-(hexan-3-yl)-3-methyl-4- (methylthio)benzamide \=o A55 O----1 r\ 44 0,12 a methyl 2-(3-chloro-4-methoxybenzamido)hexanoate 21

<img img-format="tif" img-content="drawing" file="IL173552AD000214.tif" id="idf0014" />

Table 1 - Umami Amides Compound No. Compound Umami ECso (uM) Ecjo ratio (vs. MSG) (uM) A56 0 / N-(hexan-3-yl)-3,4-imethylbenzainide 0.14 A57 (R)-methyl 4-methyl-2-(4-vi nylbenzamido)pentanoate 0.18 A58 'yM- 4 -methoxy-3 -methyl -N-(2 -melhylpentan-3-yl)benzamide 0.2 A59 c 4-inethoxy-3-methyl-N-(2-methvl hexan-3-vl)benzamide 0.2 A60 ,όΑ (R)-methyl 2-(4-(ethylthio)benzamido) -4 -methylpentanoate 0.2 22

<img img-format="tif" img-content="drawing" file="IL173552AD000215.tif" id="idf0015" />

<img img-format="tif" img-content="drawing" file="IL173552AD000216.tif" id="idf0016" />

23

<img img-format="tif" img-content="drawing" file="IL173552AD000217.tif" id="idf0017" />

Table 1 - Umami Amides Compound No. Compound Umami ECso(uM) Ec$q ratio (vs. MSG) (uM) A66 ό 53 lyj 0 4-dimethoxybenzy Tol-l-yl)isonicotii .,0 T °\ l)-3-(lH- lainide 0.29 A67 ft a' methyl 2-(3-chloro-4-methoxybenzamido)pentanoate 0.29 10.75 1 A68 4 -ethoxy-N-(heptan-4-yl)benzamide 0.32 2.62 0.3 A69 V /// (R)-methyl 4-methyl-2-(4-methylbenzamido)pentanoate 0.32 A70 _ 0 N-(heptan-4-yl)· ifluoromethyl)ben 3- zamide 0.33 24

<img img-format="tif" img-content="drawing" file="IL173552AD000218.tif" id="idf0018" />

Table 1 - Umami Amides Compound No. Compound Umami ECso («Μ) Ecjo ratio (vs. MSG) (uM) A71 4-ethyl-N-(heptan-4-yl)benzamide 0,34 A72 “'pd' 4-ethoxy-3-methyl-N-(5-methylhexan- 3-yl)benzamide 0.34 A73 χ/ (R) methylb -methyl znzamid o 0 2-(3-methoxy-4- o)-4-methylpentanoate 0.34 A74 F 3 -fluoro-N-(heptan-4-yl)-4 -methoxybenzamide 0.35 4.98 0.3 A75 / s\ γ N-(he (methylt H TV ptan-4-yl)-4- hio)benzamide 0.39 A76 0 4-methoxy-3-methyl-N-(4-phenylbu tan-2-yl)benzamide 0.4 25

<img img-format="tif" img-content="drawing" file="IL173552AD000219.tif" id="idf0019" />

<img img-format="tif" img-content="drawing" file="IL173552AD000220.tif" id="idf0020" />

26

<img img-format="tif" img-content="drawing" file="IL173552AD000221.tif" id="idf0021" />

Table 1 - Umami Amides Compound No. Compound Umami EC50 (uM) Ec50 ratio (vs. MSG) @ («Μ) A82 7 a? ° £ o' \ ex x° ° ! ethyl 4-methyl-2-(4-benzamido)pentanoate 0.51 A83 N-(heptan~ H 0 ί 4-yl)-3-methyl' benzamide 0.52 A84 OH O N-(heptan-4-yl)-2-hyd methoxybenzami roxy-3- de 0.53 A85 -0 ρΛ (R)-methyl 2-(3,5-dimethylbenzamido) -4-methylpentanoate 0.53 A86 Q v/_ methyl 2-(4-methoxy-3-methy!benzamido)-4-(methylthio)butanoate 0.53 27

<img img-format="tif" img-content="drawing" file="IL173552AD000222.tif" id="idf0022" />

Tabic 1 - Umami Amides Compound No. Compound Umami ECS0 (uM) Ecso ratio (vs. MSG) (uM) A87 -O OH )-( o 2-hydroxy-3-methoxy-N-(l,2,3,4-tetrahydronaphthalen-1 -yl)benzamide 0.54 3.8 1 A88 ψγ N-(2,4-dimethylpentan-3-yl)-3-methyl-4 -(methyl thio)benzamide 0.55 A89 ΥΊ „ αΛΥγΝγΎ 0 ® (R)-3 -chloro-4-methoxy-N-( 1 -(4-methoxyphenyl)ethyl)benzamide 0.6 2.85 1 A90 il 0 N-(heptan-4-yl)-3-methoxy benzamide 0.61 A91 —o 2=o (R)-methyl 4-methyl-2-(4-propylbenzamido)pentanoate 0.62 28

<img img-format="tif" img-content="drawing" file="IL173552AD000223.tif" id="idf0023" />

Tabic 1 - Umami Amides Compound No. Compound Umami EC50 (uM) Ecso ratio (vs. MSG) @ (uM) A92 4-ethoxy-3-methyl -N-(2 -mcthylheptan-4-yl)benzamide 0.65 A93 A/ -0 OH 2 —\ o (S)-2-hydroxy-3-methoxy-N-(l, 2,3,4-tetrahydronaphthalen-1 -yl)benzamide 0.7 5.7 1 A94 aa (R)-4-methoxy-N-(2-meihoxy-1 -phenylethyl)-3-methvlbenzamide 0.72 A95 (R)-methyi 2-(4-methoxy-3,5-dimelhylbenzamido)-4-methylpentanoate 0,74 29

<img img-format="tif" img-content="drawing" file="IL173552AD000224.tif" id="idf0024" />

Table 1 - Umami Amides Compound No. Compound Umami ECso (uM) Ecso ratio (vs. MSG) (uM) A96 ΎΊ η 0 k 4-methoxy-N-(l-(4- methoxyphenyl)propvl)-3- methyl benzamide 0.76 A97 vp / w 4-methoxy-N-(l-methoxypentan-2-yl)-3-methy Ibcnzamide 0.85 A98 \py^kZ^ Cl· 3-chloro-N-( 1 -hydroxyl-methyl pentan-2 -y 1)-4-methoxybenzamide 0.88 A99 fi /'-/'/γ·, ° (R)-methyl 4-methyl-2-(3-methylbenzain ido)pentanoate 0.89 30

<img img-format="tif" img-content="drawing" file="IL173552AD000225.tif" id="idf0025" />

Table 1 - Umami Amides Compound No. Compound Umami EC50 (uM) Ecso ratio (vs. MSG) (uM) A100 H a y γ o ' 3 -chloro-4-methoxy-N-( 1 -p-tolylethyl)benzamide 1.1 A101 OH N-(heptan-4-yl)-2 -hydroxy-4-methoxybenzamide 1.16 7.62 1 A102 ”-pk-£A 4-hydroxy-3-methyl-N-(l ,2,3,4-tetrahydronaphthalen-1 -yl)benzamide 1.32 9.49 1 A103 aA/C 1 H * 0Xt (lS}2R)-elhyl 2-(3-chloro-4-methoxybenzamido) cyclohexanecarboxylate ) 1.36 31

<img img-format="tif" img-content="drawing" file="IL173552AD000226.tif" id="idf0026" />

<img img-format="tif" img-content="drawing" file="IL173552AD000227.tif" id="idf0027" />

32

Table 2 - Umami Oxalamides

Compound No. Compound Umami ECSo (uM)' Ee» ratio (vs. MSG) _ 1 II BI Cr 0.18 N1 -(2&gt;4-dimethoxybenzyl)-N2-(2-(furan-2-yl)ethyl)oxalamide B2 0.19 /° 0 N1 -(4-ethoxy-2-methoxybenzyl)-N2-(2-(5-methylpyridin-2-yl)ethyl)oxalamide o B3 O /-' y— nh 7—ζ 0.81 N-(3 -Methyl-benzo [b] thiophen-2-ylmethy l)-N’-(2 -pyridin-2-yl-ethyl)-oxalamide B4 )—0 X—NH HN—' N—J 0 0 Nl-(2-isopropoxybenzyl)-N2-(2-(pyridin-2- yl)ethyl)oxalamide 1.22 33

Table 3 - Umami Ureas

Compound No. . IUPAC Name Umami EC50 uM EcSO ratio (vs. MSG) Con. (uM) Cl Q HN-f7 0 ' l-(2-chlorophenyl)-3-(heptan-4-yl)urea 0.37 4.95 1 C2 XX 0 l-(2,4-dichlorophenyl)-3-(l-phenYlpropyl)urea 0.49 4.52 1 C3 Cl l-(2-chlorophenyl)-3-(2-methylcyclohcxyl)urea 0.52 3.24 3 C4 HN "Λ \ 0 X 1 -(2-fluorophenyl)-3 -(heptan-4-yl)urea 0.79 12.15 3 C5 1 -(2-chlorophenyl)-3 -(1 -cyclohexylethyl)urea 0.84 9.08 1 C6 HN— O l-(4-isopropylphenyl)-3-(2-(pyridin-2-yl)ethyl)urea 0.98 C7 1 -(2-chlorophenyl)-3 -(1,2,3,4-tetrahydronaphthalen-l-yl)urea 0.99 3.68 1 34

Table 3 - Umami Ureas

Compound No. IUPAC Name Umami ECso uM Ec50 ratio (vs. MSG) Con. (uM) C8 aX , TO 1 -(2,4-dimethox&gt;phenyI)-3-(2-methylcyclohexyl)urea 1.41 2.62 0.3 C9 JY , HN-( \ l-(2-ethylphenyl)-3-(heptan-4-yl)urea 1.42 CIO l-(4-ethoxyphenyl)-3-(2-methylcyclohexyl)urea 1.51 2.1 0.3 Cll 1 -(2-fluorophenyl)-3 -(1,2,3,4-letrahydronaphthalen-l-yl)urea 1.65 4.49 1 C12 QA -oQ-^ W 0 l-(2-methoxyphenyl)-3-(2-methylcyclohexyl)urea 1.67 C13 aX 1 -(2,4-dimethoxyphenyl)-3 -(pentan-3 -yl)urea 1.72 11.87 1 35

<img img-format="tif" img-content="drawing" file="IL173552AD000228.tif" id="idf0028" />

Table 4 - Umami Acrylamides Compound No. Compound Umami EC50 (uM) Ee» ratio (vs. MSG) @ (uM) DI (E)-N-(2,4-dimethylpentan-3-yl)-3-(4- methoxyphenyl)acrylamide 0.29 3.46 1 D2 “Co (R,E)-methyl 2-(3-(4-methoxyphenyl) acrvlamido)-4-methylpentanoate 0.32 D3 0 (E)-mcthyl 2-(3-(4-methoxyphenyl) acrylamido)hexanoate 0.63 D4 Ο ι N-( 1 -Methyl-3-phenyl-propyl)-3-thiophen-2-yl-acrylamide 0.69 9.73 1 D5 (E)-N-(heptan-4-yl)-3-(4-methoxyphenyl)acrylamide 0.72 3,48 0.3 D6 0 ft-. N-( 1 -Propyl-butyl)-3-thiophen-2-yl-acrylamide 0.75 6.3 1 D7 ftJCC 0 (E)-3-(4-methoxyphenyl)-N- (pentan-3-yl)acrylamide 0.82 9.62 1 36

Table 4 - Umami Acrylamides Compound No. Compound Umami EC5o («Μ) Ee» ratio (vs. MSG) (uM) D8 ° X (R,E)-3-(4-ethoxyphenyl)-N-(l-methoxy-4-methylpentan-2 -yl)acrylamide 0,94 D9 (Z)-N-(heptan-4-yl)hex-2-enamide 0.98 DIO HN**/ t (R.E)-methyl 4-methyl-2-(3 -(thiophen-3 -yl)acrylamido)pcntanoate 1.09 Dll (R)-methyl 2-cinnamamido-4-methylpentanoate 1.17 D12 0 y-NH / (E)-4-methyl-N-(2-methylcyclohexyl) pent-2-enamide 1,28 D13 o (E)-N-sec-butyl-3-(4- ethoxyphenyl)acrylamide 1.31 2.7 0.3 37

<img img-format="tif" img-content="drawing" file="IL173552AD000229.tif" id="idf0029" />

Table 4 - Umami Acrylamides Compound No. Compound Umami ECSo (uM) Ecso ratio (vs. MSG) @ (uM) D14 vV/· 1.43 8.48 1 (E)-N-( 1 -methoxybutan-2-yl)-3-(4-methoxyphenyl)acrylamide D15 o 1.54 2.22 0.3 (E)-N-(heptan-4 -yl )-3 -(thiophen-3-yl)acrylamide D16 Z\ 'xyV 1.56 3.13 1 0 ' (E)-3-(3,4-dimethoxyphenyl)-N-(4-phenylbutan-2-yl)acrylamide 38

Table 5 - Sweet EnhanccrAmides Compound No. Compound Sweet ECso uM Umami ECso uM Umami ECso ratio El AA Cl 3 -chloro-2 -hydroxy-N-(2-methyl -1,2,3,4-tetrahydronaphtlialen-1 -yl)benzamide 0.19 E2 9/=¾ Cl (R)-3-chloro-2-hydroxy-N-(l,2,3,4-tetrahydronaphthalen-1 -yl)benzamide 0.65 E3 Cl OH /=¾ \_hOH 3-chloro-2-hydroxy-N-(5-hydroxy-l,2,3,4-tetrahydronaphthalen-1 -yl)benzamide 1.03 E4 cr oh w 3-chloro-2-hydroxy-N-(4-methyl-l,2,3,4-tetrahydronaphthalen-1 -yl)benzamide 1.61 E5 CXH TX I i 3-chloro-2-hydroxy-N-(6-methoxy-l, 2,3,4-tetrahydronaphthalen-1 -yl)benzamide 1.61 39

<img img-format="tif" img-content="drawing" file="IL173552AD000230.tif" id="idf0030" />

Tabic 5 - Sweet EnhancerAmides Compound No. Compound Sweet EC50 uM Umami EC50 uM Umami ECS0 ratio E6 3-methyl-N-(2-methyl-l,2,3,4-l-yl)isoxazole-4-ca tetra rbox te X iydr amic J onaphthalen- e 1.48 E7 0¾ Cl 3-chloro-2-hydroxy-N-( 1,2,3,4-tetrahydronaphthalen-1 -yl)benzamide 1.81 4.04 E8 90¾ OH 2,3 -dihydroxy-N-(2-methyl-1,2,3,4-tctrahydronaphthalen-1 -yl)benzamide 1.98 E9 0 2-hydroxy-N-(2-methyl-l,2,3,4 l-yl)benzam -tetri ide iliydi onaphthalen- 2.36 E10 0 c γΑ0Η OH 2,3 -dihydroxy-N-(5 -me tetrahydronaphthalen-1 tit oxy-1,2 -yl)benza ,3,4- mide 2.44 40

<img img-format="tif" img-content="drawing" file="IL173552AD000231.tif" id="idf0031" />

<img img-format="tif" img-content="drawing" file="IL173552AD000232.tif" id="idf0032" />

41

Tabic 5 - Sweet EnhanccrAmides

Compound No. Compound Sweet EC50 uM Umami EC50 uM Umami EC50 ratio E17 s P OP u oh OH (R)-2}3 -dihydroxy-N-( 1,2,3,4-tetrahydronaphthalen-l-yl)benzamide 3.13 E18 Ό 2,5-dihyi tetrahydi p XT iroxy-N-(5-methoxy-1,2,3,4-onaphthalen-1 -yl)benzamide 3.38 E19 CP F (S)-3-fluoro-2-methyl-N-(l,2,3,4-tetrahydronaphthalen-1 -yl)bcnzamide 3.57 E20 pc ΐ 1 (S)-3-chloro-2,6-dimethoxy-N-(l,2,3,4-tetrahydronaphthalen-1 -yl)benzamide 4.13 E21 V Br (R)-5-bromo-I aXi H XA ^-(1,2,3,4-tetrahydronaphthalen-1 -yl) nicotinamide 4.19 42

<img img-format="tif" img-content="drawing" file="IL173552AD000233.tif" id="idf0033" />

Table 5 - Sweet Enhancer Ami des Compound No. Compound Sweet ECso uM Umami ec50 uM Umami ECgo ratio E22 c/k Cl (R)-3-chloro-N-( 1,2,3,4-tetrahydronaphthalen- 1 -yl)benzamide 4.52 E23 (/=¾ F (R)-3 -fluoro-N-( 1,2,3,4-tetrahydronaphthalen-1 -yl)benzamide 4.86 E24 Τί/ό (R)-2,5-dihydroxy-N-( 1,2,3,4-tetrahydronaphthalen-1 -yl)benzamide 6.04 E25 kk (R)-3 -methyl -N-( 1,2,3,4-tetrahydronaph thalen-1 -yl)isoxazole-4-carboxamide 7.79 E26 /¾ (R)-5-methyl-N-(l,2,3,4-tetrahydronaphthalen-1 -yl)isoxazole-4 -carboxamide 8.09 E27 F 0 1 2,3,5,6-tetrafluoro-4 -methyl-N-(3 -methy lbutan-2-yl)benzamide 0.14 43

Table 5 - Sweet EnhancerAmides

Compound No. Compound Sweet ECS0 uM Umami EC50 uM Umami ECso ratio E28 Α'λ F 0 1 N-(3,3-dimethylbutan-2-yl)-2J3,5i6-tetrafluoro-4- methylbenzamide 0.21 E29 r QJ F NH F+° A- N-(2-methylcyclohexy 1)-3 -(trifluoromethoxy)benzamide 0.42 E30 F 3 -chloro-5 -fluoro-N-(2-methylcyclohexyl)benzamide 0.45 E31 F O ® (R)-N-(3,3-dimethylbutan-2-yl)-2,3,5,6-telrafluoro-4- methylbenzamide 0.49 E32 Ατό 4-fluoro-N-(2-methylcyclohexyl)-3- (trifluoromethyl)benzamide 0.51 E33 'tA Cl 2,5-dichloro-N-(2-methylcyclohexyl)benzamide 0.63 44

Table 5 - Sweet EnhancerAmides

Compound No. Compound Sweet ECso uM Umami ECS0 uM Umami ECso ratio E34 A F 0 1 2,3,5,6-tetrafluoro-N-(hexan-2 -yl )-4 -methyl benzamide 0.71 E35 3,5-dichloro-2,6-dimethox v-N-(2 -methylcycl ohexyl)benzamide 0.71 E36 Αό “ 2,4,6-trimethvl-N-(2-methylcyclohexyl)benzamide 0.72 E37 Yk 3,6-dichloro-2-methoxy-N-(2-methylcyclohexyl) benzamide 0.77 E38 F 0 = (S)-N-(3)3-dimetliylbutan-2-yl)-2,3}5i6-tetrdfluoro-4-methyl benzamide 0.9 E39 QyA ci o kY 2J6-dichloro-N-(2-methylcyclohexyl)benzamide 0.91 45

<img img-format="tif" img-content="drawing" file="IL173552AD000234.tif" id="idf0034" />

Table 5 - Sweet EnhancerAmides Compound No. Compound Sweet ECS0 uM Umami ECso uM Umami ECso ratio E40 2-chloro-6-methoxy-N-(2-methylcyclohexyl) isonicotinamide 0.95 9.77 E41 F\ F -) o / \ N-((2R)-bicyclo [2.2.1J heptan-2-y1)-2,3,5,6-tetrafluoro-4-methylbenzamide 1.02 E42 Vrv N-(l -methoxybutan-2-yl)-2,4-dimethylbenzamide 1.06 E43 Υγ N-(2,3 -di methy lcyclohexyl)-2,3,5,6-letrafluoro -4-methylbenzamide 1.08 E44 JH/ 1 O O 2-chloro-N-(2,3-dimethylcyclohexyl)isonicotinamide 1.08 E45 F F N-cyclohexyl -2,3,5,6 -tetrafluoro-4 -methylbenzamide 1.13 46

Table 5 - Sweet Enhancer Amides

Compound No. Compound Sweet ECso uM Umami ECso uM Umami EC50 ratio E46 N-cyclooctyl-2,3,5,6-tetrafluoro-4-methylbenzainide 1.25 E47 F χϊογ F 0 1 (R)-2,3,5,6-tetranuoro-4-methyl-N-(3-methylbutan-2- yl)benzamide 1.25 E48 3,6 -dichloro-N-(2,3 -dimethylcycl ohexy 1)-2-methoxybenzamide 1.29 E49 N-cycloheptyl -2,4,6-trimethy Ibenzamide 1.39 E50 VY N-(2,3-dimethylcyclohexyl)-2,4,6- trimethylbenzamide 1.41 E51 γ ci x/ 3-chloro-N-(2f 3-dihydro-1 H-inden-1 -yl) -2 hydroxy benzamide 1.49 47

<img img-format="tif" img-content="drawing" file="IL173552AD000235.tif" id="idf0035" />

Table 5 - Sweet EnhancerAmides Compound No. Compound Sweet ECso uM Umami ec50 11M Umami EC5o ratio E52 2-methyl-N-(2-methyIcvclohexyl)-1-naphtliamide 1,52 E53 -- X 3-chloro-4-fluoro-N-(2-methylcyclohexyl)benzamide 1.7 E54 3,4-dichloro-N-(2-methylcyclohexyI)benzamide 1.83 10.66 E55 0 ν^Χνη 5-bromo-N-(2,3 -dimethylcyclohexyl) nicoti namide 1.89 E56 C'X? w 2-chloro-N-(2-methylcyclohexyl)isonicotinamide 1.92 2.08 E57 XVb ci ο 2-chloro-3-methyl-N-(2-methylcyclohexyl)benzamide 1.95 E58 F F N-cyclopentyl-2i3,5,6-tetrafiuoro-4-methylbcnzamide 2.23 48

Table 5 - Sweet Enhancer Amides

Compound No. Compound Sweet ί ec50 uM Umami ec50 uM Umami EC50 ratio E59 N-(2-tnethylcyclohexyl)-3- (triiluoromethyl)benzamide 2.34 2.07 E60 ίθΤΟ. 4-fluoro-N-(4-methylcyclohexyl)-3- (trifluoromethyl)benzainide 2.37 E61 2-fluoro-N-(2-methylcyclohexyl)-3- (trifluoromethyl)benzamide 2,4 E62 0 ϊσ 5-bromo-N-(2-methylcyclohexyl)nicotinamide 2.42 E63 2)3-dimethyl-N-(2-methylcyclohexyl)benzamide 2.6 E64 2,6-dichloro-N-(2,3 -dimethylcycl ohexyl)benzamide 2.77 E65 0V/ KD 2-fluoro-N-(2-methylcyclohexyl)isonicotinamide 2.83 49

Table 5 - Sweet Enhancer Amides

Compound No. Compound Sweet ECso uM Umami EC50 uM Umami EC50 ratio E66 N-cyclohexyl -2,4,6-trimethylbenzamide 2.86 E67 OH 2 -hydroxy-4 -methyl-N-(4 -methylcyclohexyl)benzamide 2.98 E68 N-(heptan-4-yl)-3-(trifluoromethyl)benzamide 3.03 0.33 E69 F 0 2,3,5,6-tetrafluoro-N-isobutyl-4-metltylbenzamide 3.19 E70 F 0 ' 2,3,5,6-tetrafluoro-4-methyl-N-(5-methylhexan-2- yl)benzamide 3.2 E71 N-(2-metliylcyclohexyl)benzo[c][l,2)5]oxadiazole-5- carboxamide 3.33 E72 °H 0 Οχ, 2 -hydroxy-3 -methoxy-N-(4-methylcyclohexyl)benzamide 3.35 50

<img img-format="tif" img-content="drawing" file="IL173552AD000236.tif" id="idf0036" />

Table 5 - Sweet EnhancerAmides Compound No. Compound Sweet EC50 uM Umami EC50 uM Umami EC50 ratio E73 HN-( I ) "Hr Thiophene-2-carboxylic acid (1,3,3-trimethyl-bicyclo[2.2. l]hept-2-yl)-amide 3.36 E74 'pn'dr F N-(2,3-dimethylcyclohexyl)-2- (perfluorophenyl)acetamide 3.62 E75 ci o k^ 2,3-dichloro-N-(pentan-3-yl)benzamide 3.78 E76 2,3-dichloro-N-(2,3-dimelhylcyclohexyl)benzaiiiide 3.99 E77 N-(2,3 -di methylcyclohexyl)-2,5 -difluorobenzamide 4.11 E78 s'·"""" 4,5-Dichloro-isothiazole-3-carboxylic acid (2-methyl -cyclohexyl)-amide 4,24 8.51 51

Table 5 - Sweet EnhancerAmides

Compound No. Compound Sweet ECso uM Umami ECso uM Umami ECso ratio E79 γγ N-(2,4-dimethylpentan-3-yl)-2,6- dihydroxybenzamide 4.28 E80 3-chloro-2-methyl-N-(2-methylcyclohexyl)benzamide 4.29 E81 A 3J4-difluoro-N-(2-methvlcyclohexyl)benzamide 4.37 6.98 E82 3,5-dimethyl-N-(2-methylcyclohexyl)benzamide 4.48 E83 N-(4-ethoxyphenethyl)-1 -methyl-lH-pyrazole-5-carboxamide 4.68 E84 Y'tS /° 0 kk 3,6-dichloro-N-(2-fluorophenyl)-2-methoxy benzamide 0.83 16.51 52

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Table 5 - Sweet EnhancerAmides Compound No. Compound Sweet EC50 uM Umami EC5o uM Umami EC50 ratio E85 =%¾ N-(2 -Chloro-4,6-di methoxy-phenyl)-3 -trifluoromethyl-benzamide 1.42 E86 Cl 3,5-dichloro-N-(2,4-diincth_vlpheriyl)-4- methoxybenzamide 1.48 E87 'Xj F3-C1 I :ιΛΛΛ iloro-4 -fluoro-N-(5 -trifiuoromet 1,3,4Jthiadiazol-2-yl)-benzamid&lt; -F hyl- 1.55 E88 =3¾ Cl 3,5-dichloro-4-metlioxy-N-o-tolylbenzamide 1.84 E89 I OH O &lt; Cl 5-Chloro-N-(2,4-difluoro-phenyl)-2 -hydroxy-benzamide 2,56 E90 r 2,4-Dichloi ct ο ΛΑ-Α3 AA ii N o-N-(2-cyano-3-fluoro-phenyl)- ^"F benzamide 2.71 53

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Table 5 - Sweet EnhancerAmides Compound No. Compound Sweet ec50 uM Umami ECS0 uM Umami ec50 ratio E91 Cl o 71 2f6-Dichloro-N-(4-cyano-phenyl)- benzamide 2.74 E92 O J y 4-chloro-N-(2,4-dimethylphenyl)-3-methyIbenzamide 2.74 E93 ΡΑ4ΑΞΗ z\ 3,5 -di chloro-4-methoxv-N-(4 -methoxyphenvl)benzainide 3.24 E94 nA /° 3-chloro-N-(2I4-dimethoxyphenyl)-4- fluorobenzamide 3.56 E95 0 5-Cyano-2,4-&lt; Α&lt;ά H 0 dim ethyl-6-methyl sulfa nicotinamide a' nyl-N-phenyl- 3.58 E96 δ i^V_ 3.73 AA N-(4-tert-E aa&gt; H lutyl-thiazol-2-yI)-isonicotin amide 54

Table 5 - Sweet Enhancer Amides

Compound No. Compound Sweet ECso uM Umami ec50 uM Umami ECgo ratio E97 Ά7 3,6-Dichloro-N-(2,4 -dimethyl -phenyl )-2-methoxy-benzamide 4.25 E98 N-(3-ethylphenyl)-2-methoxy-6-methylbenzamide 4.63 E99 oc-rk' N-(4-bromo-2,6-dimethylphenyl)isoindoline-2- carboxamide 0.93 E100 )=\ O' N-(2 -methyl -4-nitrophenyl )isoindol ine-2 -carboxamide 1,3 E101 CC-T+ N-(2,4-difluorophenyl)isoindoline-2-carboxamide 1.37 E102 0¾ η ι N-(2-methyl-3-nitrophenyl)isoindoline-2- carboxamide 2.01 E103 F. F 00Ν-ξ~0- N-(2,3,4-trifluorophenyI)isoi ndol ine-2 -carboxamide 2.58 55

Table 5 - Sweet Enhancer Am ides

Compound No. Compound Sweet ec50 uM Umami EC50 uM Umami EC50 ratio E104 N-p-toIyIisoindoline-2-carboxamide 3.05 E105 ΟΡ-ΧΑ N-(4-chlorophenyl)isoi ndol ine-2-carboxamide 3.4 E106 A CC-A N-(2 -chlorophenyl)isoi ndol ine-2 -carboxamide 3.85 E107 OopAc N-(2,4-dichloropheny l)i soi ndoline-2 -carboxamide 4.15 E108 N-(4 -methoxyphenyl)isoindol ine-2 -carboxamide 4.99 E109 (ζχγΧο N-(2,4-diclilorophenyl)-3&gt;4-dihydroisoquinoline-2( IH) -carboxamide 2.34 E110 N-(2-cyanophenyl)-3,4-dihydroisoquinoline-2(lH)- carboxamide 2,5 Ell 1 N-p-t olyl-3,4 -dihydroisoquinol ine-2 (1H) -carboxamide 4.27 56

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Table 5 - Sweet EnhancerAmides Compound No. Compound Sweet ECso uM Umami EC50 uM Umami ECjo ratio E112 ' — vT CYO N-(3 -chloro-2-metliylphenyl)-3,4-dihydroisoquinoline-2( 1 H)-carboxamide 4.33 £113 / 0 QjqYY N-(2,4-dimethoxyphenyI)-3,4-dihydroisoquinoline-2(1 H)-carboxamide 4.44 57

Also, as supported by experimental data in the examples, it has been shown that cell lines which co-express T1R1/T1R3 or T1R2/T1R3 respectively respond to umami or sweet taste stimuli and a quantitative dose-responsive manner which further supports a conclusion that specific binding to the T1R1/T1R3 and T1R2/T1R3 receptor can be used to define receptor agonists and antagonists, e.g., MSG substitutes, umami blockers, novel artificial and natural sweeteners, and sweet blockers.

Also, as supported by data in experimental examples, it has been shown that the sweet taste blocker lactisole inhibits both the T1R2/T1R3 sweet receptor and the T1R1/T1R3 umami taste receptor. Compounds are provided herein that enhance, mimic, modulate or block sweet or umami taste. The fact that lactisole inhibits both the T1R1/T1R3 and T1R2/T1R3 receptors suggests that these receptors may share a common subunit which is bound by lactisole and potentially other taste modulators. Therefore, this shows that some compounds which enhance, mimic, modulate or block sweet taste can have a similar effect on umami taste or vice versa.

Further, as supported by data in experimental examples, it has been demonstrated that cell lines which stably co-express TIRs, i.e. T1R1/T1R3 or T1R2/T1R3, when assayed by automated fluorescence imaging very effectively respond to various sweet and umami taste stimuli, i.e. at magnitudes substantially greater than transiently transfected cells. Thus, these cell lines are especially well suited for use in high throughput screening assays for identifying compounds that modulate, block, mimic or enhance sweet or umami taste. However, the invention also encompasses assays that utilize cells that transiently express a T1R or combination thereof.

Moreover, while the application contains data demonstrating that some TIRs act in combination, particularly T1R1/T1R3 and T1R2/T1R3, and that such receptor combinations may be used in assays, preferably high throughput assays, it should be noted that the subject invention also encompasses assays that utilize T1R1, T1R2 and T1R3 alone or in combination with other proteins, e.g., other GPCRs.

There are differences in human and rodent sweet taste in terms of the ligand specificity, G protein coupling efficiency, as well as sensitivity to inhibitors. The species differences in T1R ligand specificity can be utilized to demonstrate that the sweet taste receptor indeed functions as a heteromeric complex, and that there is more than one ligand binding site on the receptor. Furthermore, a functional link between 58 the sweet and umami receptors mediated by T1R3 has been shown (Example 16).

Both human and rat sweet receptors can efficiently couple to a chimeric Gal 5 with the C-terminal tail sequence from Guj] (Gtti5/ii) For example, human but not rat T1R2/T1R3 selectively responds to a group of sweeteners, including aspartame, neotame, and cyclamate. This is consistent with taste physiology data. These differences in agonist specificity can be utilized to map their binding sites on the receptor. A chimeric T1R can be generated between human and rat genes, with a junction immediately before the transmembrane domain. Each T1R chimera therefore consists of two halves, the N-terminal extracellular domain, and the C-terminal transmembrane and intracellular domain, from different species. For example, a chimeric T1R2, termed T1R2-R, has a sequence from the N-terminus of human T1R2 fused to rat T1R2 C-terminal sequence. Responses to these chimeras can then be tested (Figure 22).

Novel compounds and novel flavor, tastants, and sweet enhancers were discovered in the chemistry series of amide derivatives. The amide compounds also comprise certain sub-classes of amide derivatives or classes of derivatives related to amides, such as for example ureas, urethanes, oxalamides, acrylamides, and the like. These compounds, when used together with sucrose or alone, increase a response in vitro and concomitant increase in sweet perception in human tasting. These compounds enhance other natural and synthetic sweet tastants. Examples of these compounds are listed in Table 5.

In one embodiment, the invention provides novel compounds, flavorants, tastants, flavor enhancers, taste enhancers, flavor modifying compounds, and/or compositions containing them.

In a more specific embodiment, the invention provides novel sweet flavorants, sweet tastants, sweet taste enhancers, and sweet taste modifiers and compositions containing them.

More particularly, in another embodiment, the invention is directed to compounds that modulate, induce, enhance, or inhibit natural or synthetic sweet tastants, e.g, naturally occurring and synthetic sweeteners.

In another embodiment, the invention provides compositions, preferably compositions suitable for human or animal consumption, containing at least one compound of the invention. These compositions include foods, beverages and 59

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medicinals, and food additives which when added to foods, beverages or medicinals modulate the flavor or taste thereof, particularly by enhancing the sweet taste thereof.

Another embodiment of the invention is directed to use of a compound of the invention to modulate the sweet taste of a desired food, beverage or medicinal, which 5 composition may comprise one or more other compounds that elicit a sweet taste.These compounds, when they were used together with naturally occurring and synthetic sweeteners, not only increased a response in vitro but also intensified the sweet and other flavor or taste perceptions in human tasting. These specific compounds, when they were used together with sweet tastants, such as naturally occurring and synthetic 10 sweeteners, not only increased the T1R2/T1R3 response in vitro but also intensified the sweet taste and other flavor or taste perceptions in human tasting,

Novel compounds and novel flavor, tastant, and umami enhancers and tastants such as amides, ureas, amino-amides, amido-amides, and β-lactams are also disclosed herein. These compounds, when used together with MSG or alone, increase a response 15 in vitro and the umami perception in human tasting. These compounds also enhance other natural and synthetic umami tastants. Examples of these compounds are listed in Tables 1-4.

In one embodiment, the invention provides novel compounds, flavorants, tastants, flavor enhancers, taste enhancers, flavor modifying compounds, and/or 20 compositions containing them.

In a more specific embodiment, the invention provides novel umami flavorants, umami tastants, umami taste enhancers, and umami taste modifiers and compositions containing them.

More particularly, in another embodiment, the invention is directed to 25 compounds that modulate (induce, enhance or inhibit) natural or synthetic umami tastants, e.g., monosodium glutamate (MSG).

In another embodiment, the invention provides compositions, preferably compositions suitable for human or animal consumption, containing at least one compound of the invention. These compositions include foods, beverages and 30 medicinals, and food additives which when added to foods, beverages or medicinals modulate the flavor or taste thereof, particularly by enhancing the umami taste thereof.

Another embodiment of the invention is directed to use of a compound of the invention to modulate the umami taste of a desired food, beverage or medicinal, which 60 composition may comprise one or more other compounds that elicit a umami taste, e.g., MSG. These compounds, when they were used together with MSG, not only increased a response in vitro but also intensified the umami and other flavor or taste perceptions in human tasting. These specific compounds, when they were used together with umami tastants, such as MSG, not only increased the T1R1/T1R3 response in vitro but also intensified the umami taste and other flavor or taste perceptions in human tasting. Some of the compounds, when they were tasted alone, elicited human perception of umami.

Compounds defined by specific binding to specific receptors using the present T1R assays can be used to modulate the taste of foods and beverages.

Suitable assays described in further detail infra include by way of example wholecell assays and biochemical assays, including direct-binding assays using one of a combination of different T1R receptors, chimeras or fragments thereof, especially fragments containing N-terminal ligand-binding domains. Examples of assays appropriate for use in the invention are described in greater detail infra and are known in the GPCR field.

Assays can be designed that quantitate the binding of different compounds or mixtures of compounds to TIRtaste receptors or T1R taste receptor combinations or T1R receptors expressed in combination with other heterologous (non-TIR) proteins, e.g. other GPCRs, or that quantitate the activation of cells that express T1R taste receptors. This can be effected by stably or transiently expressing taste receptors in heterologous cells such as HEK-293, CHO and COS cells. Thus, this physicochemical characteristic of the compounds is used to define a genus of compound that share this characteristic.

The assays will preferably use cells that also express (preferably stably) a G protein such as Gal 5 or Gal 6 or other promiscuous G proteins or G protein variants, or an endogenous G protein. In addition, Gpand Gy proteins may also be expressed therein.

The effect of a compound on sweet or umami taste using cells or compositions that express or contain the above-identified receptors or receptor combinations may be determined by various means including the use of calcium-sensitive dyes, voltage-sensitive dyes, cAMP assays, direct binding assays using fluorescently labeled ligands 61

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or radioactive ligands such as 3H-glutamate, or transcriptional assays (using a suitable reporter such as luciferase or beta-lactamase).

Assays that may be utilized with one or more TIRs according to the invention include by way of example, assays that utilize a genetic selection for living cells; assays 5 that utilize whole cells or membrane fragments or purified T1R proteins; assays that utilize second messengers such as cAMP and IP3, assays that detect the translocation of arrestin to the cell surface, assays that detect the loss of receptor expression on the cell surface (internalization) by tested ligands, direct ligand-binding assays, competitive-binding assays with inhibitors, assays using in vitro translated protein, assays that 10 detect conformational changes upon the binding of a ligand (e.g., as evidenced by proteolysis, fluorescence, or NMR), behavioral assays that utilize transgenic nonhuman animals that express a T1R or T1R combination, such as flies, worms, or mice, assays that utilize cells infected with recombinant viruses that contain T1R genes.

Also within the scope of the invention are structure-based analyses wherein the 15 X-ray crystal structure of a T1R or T1R fragment (or combination of TIRs, or a combination of a T1R with another protein) is determined and utilized to predict by molecular modeling techniques compounds that will bind to and/or enhance, mimic, block or modulate the particular T1R receptor or receptor combination. More particularly, the invention embraces the determination of the crystal structure of 20 T1R1/T1R3 (preferably hTlRl/hTlR3) and/or T1R2/T1R3 (preferably hTlR2/hTlR3) and the use of such crystal structures in structure-based design methods to identify molecules that modulate T1R receptor activity.

The invention especially includes biochemical assays conducted using cells, e.g., mammalian, yeast, insect or other heterologous cells that express one or more full 25 length T1R receptors or fragments, preferably N-terminal domains of T1R1, T1R2 and/or T1R3. The effect of a compound in such assays can be determined using competitive binding assays, e.g., using radioactive glutamate or IMP, fluorescence (e.g., fluorescence polarization, FRET), or GTPy S binding assays. As noted, in a preferred embodiment, such assays will utilize cell lines that stably co-express

30 T1R1/T1R3 or T1R2/T1R3 and a suitable G protein, such as Gau. Other appropriate G proteins include the chimeric and variant G proteins disclosed in U.S. Application Serial No. 09/984,292 and 60/243,770, incorporated by reference in their entirety herein. 62

Still further, altered receptors can be constructed and expressed having improved properties, e.g., enhanced surface expression or G-protein coupling. These T1R variants can be incorporated into cell-based and biochemical assays.

It is envisioned that the present discoveries relating to human TIRs will extend to other species, e.g., rodents, pigs, monkeys, dogs and cats, and perhaps even nonmammals such as fish. In this regard, several fish T1R fragments are identified infra in Example 1. Therefore, the subject invention has application in screening for compounds for use in animal feed formulations.

The invention further includes that utilize different allelic variants of various TIRs and combinations thereof, thereby enabling the identification of compounds that elicit specific taste sensation in individuals that express those allelic variants or compounds that elicit specific taste sensations in all individuals. Such compounds can be used to make foods more generally palatable. T1R encoding nucleic acids also provide valuable probes for the identification of taste cells, as the nucleic acids are specifically expressed in taste cells. For example, probes for T1R polypeptides and proteins can be used to identify taste cells present in foliate, circumvallate, and fungiform papillae, as well as taste cells present in the geschmackstreifen, oral cavity, gastrointestinal epithelium, and epiglottis. In particular, methods of detecting TIRs can be used to identify taste cells sensitive to sweet and/or umami taste stimuli or other taste stimuli representing other taste modalities. For example, cells stably or transiently expressing T1R2 and/or T1R3 would be predicted from the work herein to be responsive to sweet taste stimuli. Similarly, cells expressing T1R1 and/or T1R3 would be predicted to be responsive to umami taste stimuli. The nucleic acids encoding the T1R proteins and polypeptides of the invention can be isolated from a variety of sources, genetically engineered, amplified, synthesized, and/or expressed recombinantly according to the methods disclosed in WO 00/035374, which is herein incorporated by reference in its entirety. A listing of TIRs that may be expressed according to the invention are provided in the Examples. However, it should be emphasized that the invention embraces the expression and use of other specific TIRs or fragments, variants, or chimeras constructed based on such T1R sequences, and particularly TIRs of other species.

As disclosed, an important aspect of the invention is the plurality of methods of screening for modulators, e.g, activators, inhibitors, stimulators, enhancers, agonists, 63 and antagonists, of these taste-cell-specific GPCRs. Such modulators of taste transduction are useful for the modulation of taste signaling pathways. These methods of screening can be used to identify high affinity agonists and antagonists of taste cell activity. These modulatory compounds can then be used in the food industry to customize taste, e.g., to modulate the sweet and/or umami tastes of foods.

This invention rectifies the previous lack of understanding relating to sweet and umami taste as it identifies specific TIRs and T1R receptor combinations that mediate sweet and umami taste sensation. Therefore, in general, this application relates to the inventors’ discoveries relating to the T1R class of taste-specific G-protein-coupled receptors and their specific function in taste perception and the relationship of these discoveries to a better understanding of the molecular basis of taste.

The molecular basis of sweet taste and umami taste - the savor of monosodium glutamate - is enigmatic. Recently, a three-member class of taste-specific G-protein-coupled receptors, termed TIRs, was identified. Overlapping T1R expression patterns and the demonstration that the structurally related GAB Ab receptor is heterodimeric suggest that the TIRs function as heterodimeric taste receptors. In the examples infra, the present inventors describe the functional co-expression of human T1R1, T1R2, and T1R3 in heterologous cells; cells co-expressing T1R1 and T1R3 are activated by umami taste stimuli; cells co-expressing T1R2 and T1R3 are activated by sweet taste stimuli. T1R1/T1R3 and T1R2/T1R3 activity correlated with psychophysical detection thresholds. In addition, the 5’-ribonucleotide IMP was found to enhance the T1R1/T1R3 response to glutamate, a synergism characteristic of umami taste. These findings demonstrate that specific TIRs and particularly different combinations of the TIRs function as sweet and umami taste receptors.

Human perception of bitter, sweet, and umami is thought to be mediated by G-protein-coupled receptors (Lindemann, B., Physiol. Res. 76:718-66 (1996)). Recently, evaluation of the human genome revealed the T2R class of bitter taste receptors (Adler et al., Cell 100:613-702 (2000); Chandrasgekar et al., Celt 100:703-11 (2000); Matsunami et al., Nature 404; 601-604 (2000)) but the receptors for sweet and umami taste have not been identified. Recently, another class of candidate taste receptors, the TIRs, was identified. The TIRs were first identified by large-scale sequencing of a subtracted cDNA library derived from rat taste tissue, which identified T1R1, and subsequently by TIRl-based degenerate PCR, which led to the identification of T1R2 64 (Hoon et al., Cell 96:541-551 (1999)). Recently, the present inventors and others identified a third and possibly final member of the T1R family, T1R3, in the human genome databank (Kitagawa et al., Biochem Biophys. Res Commun, 283(1): 236-42 (2001); Max et al., fiat. Genet. 28(1): 58-63 (2001); Sainz et al., J. fieurochem. 77(3): 896-903 (2001); Montmayeur et al., fiat fieurosci. 4, 492-8. (2001)). Tellingly, mouse T1R3 maps to a genomic interval containing Sac, a locus that influences sweet taste in the mouse (Fuller et al., J. Hered 65:33-6 (1974); Li et al., Mamm. Genome 12:13-16 (2001)). Therefore, T1R3 was predicted to function as a sweet taste receptor. Recent high-resolution genetic mapping studies have strengthened the connection between mouse T1R3 and Sac (Fuller T.C., J. Hered. 65(1): 33-36 (1974); Li et al., Mammal. Genome 12(1): 13-16(2001)).

Interestingly, all C-family receptors that have been functionally expressed thus far - metabotropic glutamate receptors, the GABAb receptor, the calcium-sensing receptor (Conigrave, A. D., Quinn, S. J. &amp;amp; Brown, Ε. M., Proc Natl Acad Sci USA 97, 4814-9. (2000)), and a fish olfactory receptor (Speca, D. J. et al., fieuron 23, 487-98. (1999)) - have been shown to be activated by amino acids. This common feature raises the possibility that the TIRs recognize amino acids, and that the TIRs may be involved in the detection of glutamate in addition to sweet-tasting amino acids. Alternatively, a transcriptional variant of the mGluR4 metabotropic glutamate receptor has been proposed to be the umami taste receptor because of its selective expression in rat taste tissue, and the similarity of the receptor-activation threshold to the glutamate psychophysical detection threshold (Chaudhari et al., fiat, fieurosci. 3:113-119 (2000)) This hypothesis is difficult to reconcile with the exceedingly low expression level of the mGluR4 variant in taste tissue, and the more or less unaltered glutamate taste of mGluR4 knockout mice (Chaudhari and Roper, Ann. fi.Y. Acad. Sci. 855:398-406 (1998)). Furthermore, the taste variant is structurally implausible, lacking not only the majority of the residues that form the glutamate-binding pocket of the wild-type receptor, but also approximately half of the globular N-terminal glutamate-binding domain (Kunishima et al., Nature 407:971-7 (2000)).

Comparative analysis of T1R expression patterns in rodents has demonstrated that T1R2 and possibly T1R1 are each coexpressed with T1R3 (Hoon et al., Cell 96:541-51 (1999); Kitagawa et al., Biochem Biophy. Res. Commun. 283:236-242 (2001); Max et al., fiat. Genet 28:58-63 (2001); Montmayeur el al., fiat fieurosci 65

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4:492-8 (2001); Sainz et al., J. Neurochem 77:896-903 (2001)). Furthermore, dimerization is emerging as a common theme of C-family receptors: the metabotropic glutamate and calcium-sensing receptor are homodimers (Romomano et al., J. Biol. Chem. 271:28612-6 (1996); Okamoto et al., J. Biol. Chem. 273: 13089-96 (1998); Han 5 et al., J. Biol. Chem. 274:100008-13 (1999); Bai et at, J. Biol. Chem. 273:23605-10 (1998)), and the structurally related GAB Ab receptor is heterodimeric (Jones et al., Nature 396:674-9 (1998); Kaupmann et al., Nature 396:683-687 (1998); White et al., Nature 396: 679-682 (1998); Kuner et al., Science 283:74-77 (1999)). The present inventors have demonstrated by functional coexpression of TIRs in heterologous cells 10 that human T1R2 functions in combination with human T1R3 as a sweet taste receptor and that human T1R1 functions in combination with human T1R3 as an umami taste receptor.

The discoveries discussed herein are especially significant, as previously the development of improved artificial sweeteners has been hampered by the lack of assays 15 for sweet taste. Indeed, the five commonly used commercial artificial sweeteners, all of which activate hTlR2/hTlR3, were discovered serendipitously. Similarly, other than sensory testing, a laborious process, there is no assay for identifying compounds that modulate umami taste. These problems are now alleviated because, as established by experimental results discussed infra, the human sweet and umami receptors have been 20 identified, and assays for these receptors have been developed, particularly assays that use cells that stably express a functional T1R taste receptor, i.e. the sweet or umami taste receptor.

Based thereon the invention provides assays for detecting and characterizing taste-modulating compounds, wherein T1R family members act, as they do in the taste

25 bud, as reporter molecules for the effect on sweet and umami taste of taste-modulating compounds. Particularly provided and within the scope of the invention are assays for identifying compounds that modulate, mimic, enhance and/or block individually, sweet and umami tastes. Methods for assaying the activity of GPCRs, and especially compounds that affect GPCR activity are well known and are applicable to the T1R 30 family member ofthe present invention and functional combinations thereof. Suitable assays have been identified supra. 66

The invention also provides compounds that bind T1R1, T1R2, T1R3, T1R2/T1R3 or T1R1/T1R3, or any fragment, portion, or subunit thereof, as disclosed throughout.

In particular, the subject GPCRs can be used in assays to, e.g., measure changes in ligand binding, ion concentration, membrane potential, current flow, ion flux, transcription, receptor-ligand interactions, second messenger concentrations, in vitro and in vivo. In another embodiment, T1R family members may be recombinantly expressed in cells, and the modulation of taste transduction via GPCR activity may be assayed by measuring changes in Ca2" levels and other intracellular messages such as cAMP, cGMP, or IP3.

In certain assays, a domain of a T1R polypeptide, e.g., an extracellular, transmembrane, or intracellular domain, is fused to a heterologous polypeptide, thereby forming a chimeric polypeptide, e.g, a chimeric protein with GPCR activity.

Particularly contemplated is the use of fragments of T1R1, T1R2 or T1R3 containing the N-terminal ligand-binding domain. Such proteins are useful, e.g, in assays to identify ligands, agonists, antagonists, or other modulators of T1R receptors. For example, a T1R polypeptide can be expressed in a eukaryotic cell as a chimeric receptor with a heterologous, chaperone sequence that facilitates plasma membrane trafficking, or maturation and targeting through the secretory pathway. The optional heterologous sequence may be aPDZ domain-interacting, peptide, such as a C-terminal PDZIP fragment (SEQ ID NO 1). PDZIP is an ER export signal, which, according to the present invention, has been shown to facilitate surface expression of heterologous proteins such as the T1R receptors described herein. More particularly, in one aspect of the invention, PDZIP can be used to promote proper targeting of problematic membrane proteins such as olfactory receptors, T2R taste receptors, and the T1R taste receptors described herein.

Examples of such chimeric receptors include trans-species receptors. Any combination of receptor subunits from various species can be used together to form a chimeric receptor, which can then be used to identify tastants, for example. Therefore, contemplated herein is a chimeric T1R2/T1R3 receptor comprising a human T1R2 subunit and a rat T1R3 subunit. Also contemplated is a chimeric T1R2/T1R3 receptor comprising, a rat T1R2 subunit and a human T1R3 subunit. Also contemplated is a chimeric T1R2 receptor subunit comprising, a human extracellular domain, a rat 67

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transmembrane domain and a rat intracellular domain (SEQ ID NOS: 16 and 17, for example). Also contemplated is chimeric T1R3 receptor subunit comprising, a rat extracellular domain, a human transmembrane domain and a human intracellular domain (SEQ ID NOS: 18 and 19, for example.) 5 Such chimeric T1R receptors can be expressed in any eukaryotic cell, such as HEK-293 cells. Preferably, the cells contain a G protein, preferably a promiscuous G protein such as Gais or Gaie or another type of promiscuous G protein capable of linking a wide range of GPCRs to an intracellular signaling pathway or to a signaling protein such as phospholipase C. Activation of such chimeric receptors in such cells 10 can be detected using any standard method, such as by detecting changes in intracellular calcium by detecting FURA-2 dependent fluorescence in the cell. If preferred host cells do not express an appropriate G protein, they may be transfected with a gene encoding a promiscuous G protein such as those described in U.S. Application Serial No. 60/243,770, U.S. Application Serial No. 09/984,297, filed 15 October 29, 2001, and U.S. Application Serial No. 09/989,497 filed November 21, 2001 which are herein incorporated by reference in its entirety.

Additional methods of assaying for modulators of taste transduction include in vitro ligand-binding assays using: T1R polypeptides, portions thereof, z'.e, the extracellular domain, transmembrane region, or combinations thereof, or chimeric 20 proteins comprising one or more domains of a T1R family member; oocyte or tissue culture cells expressing T1R polypeptides, fragments, or fusion proteins; phosphorylation and dephosphorylation of T1R family members; G protein binding to GPCRs; ligand-binding assays; voltage, membrane potential and conductance changes; ion flux assays; changes in intracellular second messengers such as cGMP, cAMP and 25 inositol triphosphate (IP3); and changes in intracellular calcium levels.

Further, the invention provides methods of detecting T1R nucleic acid and protein expression, allowing investigation of taste transduction regulation and specific identification of taste receptor cells. T1R family members also provide useful nucleic acid probes for paternity and forensic investigations. T1R genes are also useful as 30 nucleic acid probes for identifying taste receptor cells, such as foliate, fungiform, circum val late, geschmackstreifen, and epiglottis taste receptor cells. T1R receptors can 68

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also be used to generate monoclonal and polyclonal antibodies useful for identifying taste receptor cells.

Functionally, the T1R polypeptides comprise a family of related seven transmembrane G protein-coupled receptors, which are believed to be involved in taste 5 transduction and may interact with a G protein to mediate taste signal transduction (see, e.g., Fong, Cell Signal, 8:217 (1996); Baldwin, Curr. Opin. Cell Biol., 6:180 (1994)). Structurally, the nucleotide sequences of T1R family members encode related polypeptides comprising an extracellular domain, seven transmembrane domains, and a cytoplasmic domain. Related T1R family genes from other species share at least about 10 50%, and optionally 60%, 70%, 80%, or 90%, nucleotide sequence identity over a region of at least about 50 nucleotides in length, optionally 100, 200, 500, or more nucleotides in length to the T1R nucleic acid sequences disclosed herein in the Examples, or conservatively modified variants thereof, or encode polypeptides sharing at least about 35 to 50%, and optionally 60%, 70%, 80%, or 90%, amino acid sequence 15 identity over an amino acid region at least about 25 amino acids in length, optionally 50 to 100 amino acids in length to a T1R polypeptide sequence disclosed infra in the Examples conservatively modified variants thereof.

Several consensus amino acid sequences or domains have also been identified that are characteristic of T1R family members. For example, T1R family members

20 typically comprise a sequence having at least about 50%, optionally 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95-99%, or higher, identity to T1R consensus sequences 1 and 2 (SEQ ED NOs. 2 and 3, respectively). These conserved domains thus can be used to identify members of the T1R family, by identity, specific hybridization or amplification, or specific binding by antibodies raised against a domain. T1R 25 consensus sequences include by way of example the following sequences: T1RFamily Consensus Sequence 1: (SEQ IDNO: 2) (TR)C(FL)(RQP)R(RT)(SPV)(VERKT)FL(AE)(WL)(RHG)E T1R Family Consensus Sequence 2: (SEQ ID NO: 3) (LQ)P(EGT)(NRC)YN(RE)A(RK)(CGF)(VLI)T(FL)(AS)(ML)

30 These consensus sequences are inclusive of those found in the T1R polypeptides described herein, but T1R family members from other organisms may be expected to comprise consensus sequences having about 75% identity or more to the inclusive consensus sequences described specifically herein. 69

Specific regions of the T1R nucleotide and amino acid sequences may be used to identify polymorphic variants, interspecies homologs, and alleles of T1R family members. This identification can be made in vitro, e.g., under stringent hybridization conditions or PCR (e.g., using primers encoding the T1R consensus sequences identified above), or by using the sequence information in a computer system for comparison with other nucleotide sequences. Different alleles of T1R genes within a single species population will also be useful in determining whether differences in allelic sequences control differences in taste perception between members of the population. Classical PCR-type amplification and cloning techniques are useful for isolating new TIRs, for example, where degenerate primers are sufficient for detecting related genes across species.

Typically, identification of polymorphic variants and alleles of T1R family members can be made by comparing an amino acid sequence of about 25 amino acids or more, e.g., 50-100 amino acids. Amino acid identity of approximately at least 35 to 50%, and optionally 60%, 70%, 75%, 80%, 85%, 90%, 95-99%, or above typically demonstrates that a protein is a polymorphic variant, interspecies homolog, or allele of a T1R family member, Sequence comparison can be performed using any of the sequence comparison algorithms discussed below. Antibodies that bind specifically to T1R polypeptides or a conserved region thereof can also be used to identify alleles, interspecies homologs, and polymorphic variants.

Polymorphic variants, interspecies homologs, and alleles of T1R genes can be confirmed by examining taste-cell-specific expression of the putative T1R gene or protein. Typically, T1R polypeptides having an amino acid sequence disclosed herein can be used as a positive control in comparison to the putative T1R polypeptide to demonstrate the identification of a polymorphic variant or allele of the T1R family member. The polymorphic variants, alleles, and interspecies homologs are expected to retain the seven transmembrane structure of a G protein-coupled receptor. For further detail, see WO 00/06592, which discloses related T1Rfamily members, GPCR-B3s, the contents of which are herein incorporated by reference in a manner consistent with this disclosure. GPCR-B3 receptors are referred to herein as rTIRl and mTIRl. Additionally, see WO 00/06593, which also discloses related T1R family members, GPCR-B4s, the contents of which are herein incorporated by reference in a manner consistent with this disclosure. GPCR-B4 receptors are referred to herein as rT!R2 and 70 mTlR2. As discussed previously, the invention also includes structure-based assays that utilize the x-ray crystalline structure of a T1R or T1R combination, e.g., hTlR2/hTlR3 or hTlRl/hTlR3, to identify molecules that modulate T1R receptor activity, and thereby modulate sweet and/or umami taste.

The present invention also provides assays, preferably high throughput assays, to identify molecules that enhance, mimic, block and/or modulate T1R receptors. In some assays, a particular domain of a T1R family member is used in combination with a particular domain of another T1R family member, e.g., an extracellular, transmembrane, or intracellular domain or region. In other embodiments, an extracellular domain, transmembrane region or combination thereof may be bound to a solid substrate, and used, e.g., to isolate ligands, agonists, antagonists, or any other molecules that can bind to and/or modulate the activity of a T1R polypeptide.

Various conservative mutations and substitutions are envisioned to be within the scope of the invention. For instance, it is within the level of skill in the art to perform amino acid substitutions using known protocols of recombinant gene technology including PCR, gene cloning, site-directed mutagenesis of cDNA, transfection of host cells, and in-vitro transcription. The variants could then be screened for activity.

Definitions

As used herein, the following terms have the meanings ascribed to them unless specified otherwise. “Taste cells” include neuroepithelial cells that are organized into groups to form taste buds of the tongue, e.g, foliate, fungiform, and circumvallate cells (see, e.g, Roper et al., Ann. Rev. Neurosci. 12:329-353 (1989)). Taste cells are also found in the palate and other tissues, such as the esophagus and the stomach. “T1R” refers to one or more members of a family of G protein-coupled receptors that are expressed in taste cells such as foliate, fungiform, and circumvallate cells, as well as cells of the palate, and esophagus (see, e.g, Hoon et al., Cell, 96:541-551 (1999), herein incorporated by reference in its entirety). Members of this family are also referred to as GPCR-B3 and TRI in WO 00/06592 as well as GPCR-B4 and TR2 in WO 00/06593. GPCR-B3 is also herein referred to as rTIRl, and GPCR-B4 is referred to as rTlR2. Taste receptor cells can also be identified on the basis of morphology (see, e.g, Roper, supra), or by the expression of proteins specifically 71

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expressed in taste cells. T1R family members may have the ability to act as receptors for sweet taste transduction, or to distinguish between various other taste modalities. Representative T1R sequences, including hTIRl, hTlR2 and hTlR3 are identified infra in the examples. 5 “T1R” nucleic acids encode a family of GPCRs with seven transmembrane regions that have “G protein-coupled receptor activity,” e.g, they may bind to G proteins in response to extracellular stimuli and promote production of second messengers such as IP3, cAMP, cGMP, and Ca2+ via stimulation of enzymes such as phospholipase C and adenylate cyclase (for a description ofthe structure and function 10 of GPCRs, see, e.g, Fong, supra, and Baldwin, supra). A single taste cell may contain many distinct T1R polypeptides.

The term “T1R” family therefore refers to polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have at least about 35 to 50% amino acid sequence identity, optionally about 60, 75, 80, 85, 90, 95, 96, 97, 98, or 99% amino 15 acid sequence identity to a T1R polypeptide, preferably those identified in Example 1, over a window of about 25 amino acids, optionally 50-100 amino acids; (2) specifically bind to antibodies raised against an immunogen comprising an amino acid sequence preferably selected from the group consisting of the T1R polypeptide sequence disclosed in Example 1 and conservatively modified variants thereof; (3) are encoded 20 by a nucleic acid molecule which specifically hybridize (with a size of at least about 100, optionally at least about 500-1000 nucleotides) under stringent hybridization conditions to a sequence selected from the group consisting of the T1R nucleic acid sequences contained in Example 1, and conservatively modified variants thereof; or (4) comprise a sequence at least about 35 to 50% identical to an amino acid sequence 25 selected from the group consisting of the T1R amino acid sequence identified in Example 1.

Topologically, the TIRs disclosed herein have an “N-terminal domain” also called “extracellular domain” comprising a “venus flytrap domain” and a “cysteine rich domain;” “transmembrane domains” comprising seven transmembrane regions, and 30 corresponding cytoplasmic, and extracellular loops; and a “C-terminal domain” (see, e.g, Hoon etai, Cell, 96:541-551 (1999); Buck &amp;amp; Axel, Cell, 65:175-187 (1991)). These domains have been structurally identified using methods known to those of skill in the art, such as sequence analysis programs that identify hydrophobic and 72

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hydrophilic domains (Stryer, Biochemistry, (3rd ed. 1988). Such domains are useful for making chimeric proteins and for in vitro assays of the invention, e.g, ligand binding assays. The specific binding of a compound to these structurally defined domains provides provides structural definition for the compound. 5 “Extracellular domains” therefore refers to the domains of T1R polypeptides that protrude from the cellular membrane and are exposed to the extracellular face of the cell. Such domains generally include the “N terminal domain” that is exposed to the extracellular face of the cell, and optionally can include portions ofthe extracellular loops of the transmembrane domain that are exposed to the extracellular face of the 10 cell, i.e., the loops between transmembrane regions 2 and 3, between transmembrane regions 4 and 5, and between transmembrane regions 6 and 7,

The “N-terminal domain” region starts at the N-terminus and extends to a region close to the start of the First transmembrane domain. More particularly, in one embodiment of the invention, this domain starts at the N-terminus and ends 15 approximately at the conserved glutamic acid at amino acid position 563 plus or minus approximately 20 amino acids. These extracellular domains are useful for in vitro ligand-binding assays, both soluble and solid phase. In addition, transmembrane regions, described below, can also bind ligand either in combination with the extracellular domain, and are therefore also useful for in vitro ligand-binding assays. 20 “Cysteine-rich domain” refers to the domain of the polypeptides. This conserved sequence contains several highly-conserved Cys residues that form disulphide bridges, and lies outside the cell membrane.. This region corresponds to the domain of the T1R family members and is found in all three subunits, T1R1-T1R3. The cysteine rich sequence is found in amino acids 510-566 of T1R1, 508-565 ofTlR2, and 25 512-568 or T1R3.

“Transmembrane domain,” which comprises the seven “transmembrane regions,*’ refers to the domain of T1R polypeptides that lies within the plasma membrane, and may also include the corresponding cytoplasmic (intracellular) and extracellular loops. In one embodiment, this region corresponds to the domain of T1R 30 family members which starts approximately at the conserved glutamic acid residue at amino acid position 563 plus or minus 20 amino acids and ends approximately at the conserved tyrosine amino acid residue at position 812 plus or minus approximately 10 amino acids. The seven transmembrane regions and extracellular and cytoplasmic loops 73

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can be identified using standard methods, as described in Kyte &amp;amp; Doolittle, J. Mol. Biol., 157:105-32 (1982)), or in Stryer, supra, “Cytoplasmic domains” refers to the domains of T1R polypeptides that face the inside of the cell, e.g., the “C-terminal domain” and the intracellular loops ofthe 5 transmembrane domain, e.g., the intracellular loop between transmembrane regions 1 and 2, the intracellular loop between transmembrane regions 3 and 4, and the intracellular loop between transmembrane regions 5 and 6. “C-terminal domain” refers to the region that spans the end of the last transmembrane domain and the C-terminus of the protein, and which is normally 10 located within the cytoplasm. In one embodiment, this region starts at the conserved tyrosine amino acid residue at position 812 plus or minus approximately 10 amino acids and continues to the C-terminus of the polypeptide.

The term “ligand-binding region” or “ligand-binding domain” refers to sequences derived from a taste receptor, particularly a taste receptor that substantially 15 incorporates at least the extracellular domain of the receptor. In one embodiment, the extracellular domain of the ligand-binding region may include the N-terminal domain and, optionally, portions ofthe transmembrane domain, such as the extracellular loops of the transmembrane domain. The ligand-binding region may be capable of binding a ligand, and more particularly, a compound that enhances, mimics, blocks, and/or 20 modulates taste, e.g., sweet or umami taste.

The phrase “heteromultimer” or “heteromultimeric complex” in the context of the T1R receptors or polypeptides of the invention refers to a functional association of at least one T1R receptor and another receptor, typically another T1R receptor polypeptide (or, alternatively another non-TIR receptor polypeptide), For clarity, the 25 functional co-dependence of the TIRs is described in this application as reflecting their possible function as heterodimeric taste receptor complexes. However, as discussed previously, functional co-dependence may alternatively reflect an indirect interaction. For example, T1R3 may function solely to facilitate surface expression of T1R1 and T1R2, which may act independently as taste receptors. Alternatively, a functional taste 30 receptor may be comprised solely of T1R3, which is differentially processed under the control of T1R1 or T1R2, analogous to RAMP-dependent processing of the calcium-related receptor. 74

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The phrase “functional effects” in the context of assays for testing compounds that modulate T1R family member mediated taste transduction includes the determination of any parameter that is indirectly or directly under the influence of the receptor, e.g., functional, physical and chemical effects. It includes ligand binding, 5 changes in ion flux, membrane potential, current flow, transcription, G protein binding, GPCR phosphorylation or dephosphorylation, conformation change-based assays, signal transduction, receptor-ligand interactions, second messenger concentrations (e.g, cAMP, cGMP, IP3, or intracellular Ca2+), in vitro, in vivo, and ex vivo and also includes other physiologic effects such increases or decreases of neurotransmitter or hormone 10 release.

By “determining the functional effect” in the context of assays is meant assays for a compound that increases or decreases a parameter that is indirectly or directly under the influence of a T1R family member, e.g, functional, physical and chemical effects. Such functional effects can be measured by any means known to those skilled 15 in the art, e.g, changes in spectroscopic characteristics (e.g, fluorescence, absorbency, refractive index), hydrodynamic (e.g, shape), chromatographic, or solubility properties, patch clamping, voltage-sensitive dyes, whole cell currents, radioisotope efflux, inducible markers, oocyte T1R gene expression; tissue culture cell T1R expression; transcriptional activation of T1R genes; ligand-binding assays; voltage, membrane 20 potential and conductance changes; ion flux assays; changes in intracellular second messengers such as cAMP, cGMP, and inositol triphosphate (IP3); changes in intracellular calcium levels; neurotransmitter release, conformational assays and the like. A “flavor or tastant” herein refers to a compound or biologically acceptable salt 25 thereof that induces, in a subject, the perception of smell and/or taste, which include sweet, sour, salty, bitter and umami, and others. The subject can be human, animals, and/or a biological assay, such as the ones described and cited in this application. A “flavor or taste modifier” herein refers to a compound or biologically acceptable salt thereof that modulates, including enhancing or potentiating, inhibiting, 30 and inducing, the smell and/or tastes of a natural or synthetic tastants in a subject. A “flavor or taste enhancer” herein refers to a compound or biologically acceptable salt thereof that enhances the tastes or smell of a natural or synthetic 75

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tastants, e.g., monosodium glutamate (MSG) for umami taste and fructose for sweet taste. “Umami tastant” or “umami compound” herein refers to a compound or biologically acceptable salt thereof that elicits a detectable umami taste in a subject, 5 e.g, MSG. “Sweet tastant” or “sweet compound” herein refers to a compound or biologically acceptable salt thereof that elicits a detectable sweet taste in a subject, e.g., fructose.

An “umami taste modifier” herein refers to a compound or biologically 10 acceptable salt thereof that modulates, including enhancing or potentiating, inhibiting, and inducing, the umami taste of a natural or synthetic umami tastants, e.g, monosodium glutamate (MSG) in a subject. A “sweet taste modifier” herein refers to a compound or biologically acceptable salt thereof that modulates, including enhancing or potentiating, inhibiting, and 15 inducing, the sweet taste of a natural or synthetic sweet tastants, e.g., fructose, in a subject. A “taste enhancing amount” herein refers to an amount of a compound that is sufficient to enhance the taste of a natural or synthetic tastants, e.g., monosodium glutamate (MSG) for umami taste or fructose for sweet taste. 20 “Wet Soup Category” means wet/liquid soups regardless of concentration or container, including frozen Soups. For the purpose of this definition soup(s) means a food prepared from meat, poultry, fish, vegetables, grains, fruit and other ingredients, cooked in a liquid which may include visible pieces of some or all of these ingredients. It may be clear (as a broth) or thick (as a chowder), smooth, pureed or chunky, ready- 25 to-serve, semi-condensed or condensed and may be served hot or cold, as a first course or as the main course of a meal or as a between meal snack (sipped like a beverage). Soup may be used as an ingredient for preparing other meal components and may range from broths (consomme) to sauces (cream or cheese-based soups). “Dehydrated and Culinary Food Category” means: (i) Cooking aid products 30 such as: powders, granules, pastes, concentrated liquid products, including concentrated bouillon, bouillon and bouillon like products in pressed cubes, tablets or powder or granulated form, which are sold separately as a finished product or as an ingredient within a product, sauces and recipe mixes (regardless of technology); (ii) Meal 76 solutions products such as: dehydrated and freeze dried soups, including dehydrated soup mixes, dehydrated instant soups, dehydrated ready-to-cook soups, dehydrated or ambient preparations of ready-made dishes, meals and single serve entrees including pasta, potato and rice dishes; and (iii) Meal embellishment products such as: condiments, marinades, salad dressings, salad toppings, dips, breading, batter mixes, shelf stable spreads, barbecue sauces, liquid recipe mixes, concentrates, sauces or sauce mixes, including recipe mixes for salad, sold as a finished product or as an ingredient within a product, whether dehydrated, liquid or frozen. “Beverage Category” means beverages, beverage mixes and concentrates, including but not limited to, alcoholic and non-alcoholic ready to drink and dry powdered Other examples of foods and beverages wherein compounds according to the invention may be incorporated included by way of example carbonated and non-carbonated beverages, e.g., sodas, juices, alcoholic and non-alcoholic beverages, confectionary products, e.g, cakes, cookies, pies, candies, chewing gums, gelatins, ice creams, sorbets, puddings, jams, jellies, salad dressings, and other condiments, cereal, and other breakfast foods, canned fruits and fruit sauces and the like.

Additionally, the subject compounds can be used in flavor preparations to be added to foods and beverages. In preferred instances the composition will comprise another flavor or taste modifier such as a sweet tastant.

In some instances biologically acceptable salts of the subject compounds may be used. Examples of such salts include alkali and earth metal salts, organic salts, and the like. Specific examples include potassium, sodium, calcium and magnesium salts, hydrochloric or sulfuric acid salts, ethanolamine salts, and the like. The salt will be selected such that it is biologically safe for ingestion and does adversely affect the sweet taste modulatory properties of the compound.

As used herein, the term “medicinal product” includes both solids and liquids which are ingestible non-toxic materials which have medicinal value such as cough syrups, cough drops, aspirin and chewable medicinal tablets. An oral hygiene product includes solids and liquids such as toothpaste or mouthwash. A “comestibly or medicinally acceptable carrier or excipient” is a medium that is used to prepare a desired dosage form of the inventive compound. A comestibly or medicinally acceptable carrier includes solvents, diluents, or other liquid vehicle; 77 dispersion or suspension aids; surface active agents; isotonic agents; thickening or emulsifying agents, preservatives; solid binders; lubricants and the like. “Inhibitors,” “activators,” “enhancers” and “modulators” of T1R genes or proteins are used to refer to inhibitory, activating, enhancing or modulating molecules identified using in vitro and in vivo assays for taste transduction, e.g., ligands, agonists, antagonists, and their homologs and mimetics.

Inhibitors are compounds that, e.g, bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate taste transduction, e.g, antagonists. Activators and enhancers are compounds that, e.g, bind to, enhance, stimulate, increase, open, activate, facilitate, enhance activation, sensitize, or up regulate taste transduction, e.g, agonists. Modulators include compounds that, e.g, alter the interaction of a receptor with: extracellular proteins that bind activators or inhibitor (e.g, ebnerin and other members of the hydrophobic carrier family); G proteins; kinases (e.g, homologs of rhodopsin kinase and beta adrenergic receptor kinases that are involved in deactivation and desensitization of a receptor); and arrestins, which also deactivate and desensitize receptors. Modulators can include genetically modified versions of T1R family members, e.g, with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. Such assays for inhibitors and activators include, e.g, expressing T1R family members in cells or cell membranes, applying putative modulator compounds, in the presence or absence of tastants, e.g, sweet tastants, and then determining the functional effects on taste transduction, as described above. Samples or assays comprising T1R family members that are treated with a potential enhancer, activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of modulation. Positive control samples (e.g. a sweet tastant without added modulators) are assigned a relative T1R activity value of 100%. “EC50” is defined as the amount of a compound that elicits 50% of the maximal response the compound can elicit, whether as an activator, enhancer, or modulator. A dose-dependent response curve was determined for a compound, and the compound concentration corresponding to 50% of the maximal response was derived from the curve, in one example. 78

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“ICso” is defined as the amount of a compound that elicits 50% of the maximal effect the compound can elicit as an inhibitor.

Regarding sweet tastants and enhancers, after a compound is identified, scores of their activities are given as percentage of the maximum fructose intensity (%). In 5 compound dose response, an EC50 can be calculated to reflect the potency of the compound as a sweet agonist. In the present invention, an EC50 of lower than about 100 mM is indicative of compounds that induce T1R2/T1R3 activity as a sweet agonist. Preferably, a positive hit for a sweet agonist has an EC50 value of less than about 1 mM; more preferably less than about a 10 μΜ. 10 In sweet enhancement assay experiments, a fructose dose response was run and a second fructose dose response was run with a certain amount of candidate compound at every fructose concentrations at the same time. Then, the EC50 ratio can be calculated based on the following definitions: EC50 Ratio “ EC50 (fructose)/EC5o (fructose + [Compound]) 15 wherein “[compound]” refers to the concentration of compound used to elicit (or enhance or potentiate) the fructose dose response. Those concentrations could vary from a pM to an mM, more preferred, from a low nM to μΜ. A potent sweet enhancer would have a high EC50 Ratio at a low concentration of the compound used.

In the present invention, an EC50 ratio of greater than 1 is indicative of a 20 compound that modulates (potentiates) T1R2/T1R3 activity and is an sweet enhancer. Preferably, a positive hit will have EC50 ratio values of at least 1.20, preferably ranging from at least 1.50 to 100 or even higher.

By contrast, competing agonists (those sweet tastants that bind mutually exclusively) or inhibitors always yield values of EC50 ratio less than 1, such as from 0- 25 1.

Regarding umami tastants and enhancers, scores of their activities can be given as percentage of the maximum MSG intensity (%). In compound dose response, an EC50 can be calculated to reflect the potency of the compound as umami agonist. In the present invention, an EC50 of lower than about 10 mM is indicative of compounds that 30 induce T1R1/T1R3 activity and an umami agonist. Preferably, a positive hit for an umami agonist will have EC50 values of less than about 1 mM; more preferably ranging from about a pM to about a low μΜ. 79

In enhancement assay experiments, a MSG dose response was run and a second MSG dose response was run with a certain amount of candidate compound at every MSG concentrations at the same time. Then, the EC50 ratio is calculated based on the following definitions: EC50 Ratio = EC50 (MSGj/ECso (MSG + [Compound]) wherein “[compound]” refers to the concentration of compound used to elicit (or enhance or potentiate) the MSG dose response. Those concentrations can vary from a pM to an mM, more preferred, from a low nM to μΜ. A potent umami enhancer has a high EC50 Ratio at a low concentration of the compound used.

In the present invention, an EC50 ratio of greater than 1 is indicative of a compound that modulates (potentiates) T1R1/T1R3 activity and in an umami enhancer. Preferably, a positive hit has EC50 ratio values of at least 1.20, preferably ranging from at least 1.50 to 100 or even higher.

Negative control samples (e.g. buffer without an added taste stimulus) are assigned a relative T1R activity value of 0%. Inhibition of a T1R is achieved when a mixture of the positive control sample and a modulator result in the T1R activity value relative to the positive control is about 80%, optionally 50% or 25-0%. Activation of a T1R by a modulator alone is achieved when the T1R activity value relative to the positive control sample is 10%, 25%, 50%, 75%, optionally 100%, optionally 150%, optionally 200-500%; or 1000-3000% higher.

The terms “purified,” “substantially purified,” and “isolated” as used herein refer to the state of being free of other, dissimilar compounds with which the compound of the invention is normally associated in its natural state, so that the “purified,” “substantially purified,” and “isolated” subject comprises at least 0.5%, 1%, 5%, 10%, or 20%, and most preferably at least 50% or 75% of the mass, by weight, of a given sample. In one preferred embodiment, these terms refer to the compound of the invention comprising at least 95% of the mass, by weight, of a given sample. As used herein, the terms “purified,” “substantially purified,” and “isolated,” when referring to a nucleic acid or protein, also refers to a state of purification or concentration different than that which occurs naturally in the mammalian, especially human body. Any degree of purification or concentration greater than that which occurs naturally in the mammalian, especially human, body, including (1) the purification from other associated structures or compounds or (2) the association with structures or compounds 80

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to which it is not normally associated in the mammalian, especially human, body, are within the meaning of “isolated.” The nucleic acid or protein or classes of nucleic acids or proteins, described herein, may be isolated, or otherwise associated with structures or compounds to which they are not normally associated in nature, according to a 5 variety of methods and processes known to those of skill in the art.

The term “nucleic acid” or “nucleic acid sequence” refers to a deoxy ribonucleotide or ribonucleotide oligonucleotide in either single- or double-stranded form. The term encompasses nucleic acids, i.e., oligonucleotides, containing known analogs of natural nucleotides. The term also encompasses nucleic-acid-like structures 10 with synthetic backbones (see e.g., Oligonucleotides and Analogues, aPractical

Approach, ed. F. Eckstein, Oxford Univ. Press (1991); Antisense Strategies, Annals of the N.Y. Academy of Sciences, Vol. 600, Eds. Baserga et al. (NYAS 1992); Milligan J. Med. Chem. 36:1923-1937 (1993); Antisense Research and Applications (1993, CRC Press), WO 97/03211; WO 96/39154; Mata, Toxicol. Appl. Pharmacol. 144:189-197 15 (1997); Strauss-Soukup, Biochemistry 36:8692-8698 (1997); Samstag, Antisense

Nucleic Acid Drug Dev, 6:153-156 (1996)).

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g, degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly 20 indicated. Specifically, degenerate codon substitutions may be achieved by generating, e.g, sequences in which the third position of one or more selected codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka etal., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). The term nucleic acid is used interchangeably with 25 gene, cDNA, mRNA, oligonucleotide, and polynucleotide.

The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. Tbe terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino 30 acid polymers and non-naturally occurring amino acid polymer.

The term “plasma membrane translocation domain” or simply “translocation domain” means a polypeptide domain that, when incorporated into a polypeptide coding sequence, can with greater efficiency “chaperone” or “translocate” the hybrid 81

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(“fusion”) protein to the cell plasma membrane than without the domain. For instance, a “translocation domain” may be derived from the amino terminus of the bovine rhodopsin receptor polypeptide, a 7-transmembrane receptor. However, rhodopsin from any mammal may be used, as can other translocation facilitating sequences. Thus, 5 the translocation domain is particularly efficient in translocating 7-transmembrane fusion proteins to the plasma membrane, and a protein (e.g, a taste receptor polypeptide) comprising an amino terminal translocating domain will be transported to the plasma membrane more efficiently than without the domain. However, if the N-terminal domain of the polypeptide is active in binding, as with the T1R receptors of 10 the present invention, the use of other translocation domains may be preferred. For instance, a PDZ domain-interacting peptide, as described herein, may be used.

The “translocation domain,” “ligand-binding domain”, and chimeric receptors compositions described herein also include “analogs,” or “conservative variants” and “mimetics” (“peptidomimetics”) with structures and activity that substantially 15 correspond to the exemplary sequences. Thus, the terms “conservative variant” or “analog” or “mimetic” refer to a polypeptide which has a modified amino acid sequence, such that the change(s) do not substantially alter the polypeptide’s (the conservative variant’s) structure and/or activity, as defined herein. These include conservatively modified variations of an amino acid sequence, i.e., amino acid 20 substitutions, additions or deletions of those residues that are not critical for protein activity, or substitution of amino acids with residues having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non-polar, etc.) such that the substitutions of even critical amino acids does not substantially alter structure and/or activity. 25 More particularly, “conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy 30 of the genetic code, a large number of functionally identical nucleic acids encode any given protein.

For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can 82 be altered to any of the corresponding codons described without altering the encoded polypeptide.

Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein, which encodes a polypeptide, also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid, which encodes a polypeptide, is implicit in each described sequence.

Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, one exemplary guideline to select conservative substitutions includes (original residue followed by exemplary substitution): ala/gly or ser; arg/lys; asn/gln or his; asp/glu; cys/ser; gln/asn; gly/asp; gly/ala or pro; his/asn or gin; ile/leu or val; leu/ile or val; lys/arg or gin or glu; met/leu or tyr or ile; phe/met or leu or tyr; ser/thr; thr/ser; trp/tyr; tyr/trp or phe; val/ile or leu. An alternative exemplary guideline uses the following six groups, each containing amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (I); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); (see also, e.g., Creighton, Proteins, W.H. Freeman and Company (1984); Schultz and Schimer, Principles of Protein Structure, Springer-Vrlag (1979)). One of skill in the art will appreciate that the above-identified substitutions are not the only possible conservative substitutions. For example, for some purposes, one may regard all charged amino acids as conservative substitutions for each other whether they are positive or negative. In addition, individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence can also be considered “conservatively modified variations.”

The terms “mimetic” and “peptidomimetic” refer to a synthetic chemical compound that has substantially the same structural and/or functional characteristics of the polypeptides, e.g., translocation domains, ligand-binding domains, or chimeric receptors of the invention. The mimetic can be either entirely composed of synthetic, 83 non-natural analogs of amino acids, or may be a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the mimetic’s structure and/or activity.

As with polypeptides ofthe invention which are conservative variants, routine experimentation will determine whether a mimetic is within the scope of the invention, i.e., that its structure and/or function is not substantially altered. Polypeptide mimetic compositions can contain any combination of non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond (“peptide bond”) linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like. A polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g, glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N’-dicyclohexylcarbodiimide (DCC) orN,N’-diisopropylcarbodiimide (D1C). Linking groups that can be an alternative to the traditional amide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g., -C(=O)-CH2- for -C(=O)-NH-), aminomethylene (CH2-NH), ethylene, olefin (CH=CH), ether (CH2-O), thioether (CH2-S), tetrazole (CN4), thiazole, retroamide, or ester (see, e.g, Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY (1983)). A polypeptide can also be characterized as a mimetic by containing all or some non-natural residues in place of naturally occurring amino acid residues; non-natural residues are well described in the scientific and patent literature. A “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include 32P, fluorescent dyes, electron-dense reagents, enzymes (e.g, as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g, by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide. 84

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A “labeled nucleic acid probe or oligonucleotide” is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe. 5 As used herein a “nucleic acid probe or oligonucleotide” is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.). In addition, the bases in 10 a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization. Thus, for example, probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence 15 depending upon the stringency of the hybridization conditions. The probes are optionally directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind.

By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence. 20 The term “heterologous” when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g, a promoter from one source and a 25 coding region from another source. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g, a fusion protein). A “promoter” is defined as an array of nucleic acid sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic 30 acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs 85

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from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental and developmental conditions.

An “inducible” promoter is a promoter that is active under environmental or developmental regulation. The term “operably linked” refers to a functional linkage 5 between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription ofthe nucleic acid corresponding to the second sequence.

As used herein, “recombinant” refers to a polynucleotide synthesized or 10 otherwise manipulated in vitro (e.g., “recombinant polynucleotide”), to methods of using recombinant polynucleotides to produce gene products in cells or other biological systems, or to a polypeptide (“recombinant protein”) encoded by a recombinant polynucleotide. “Recombinant means” also encompass the ligation of nucleic acids having various coding regions or domains or promoter sequences from different 15 sources into an expression cassette or vector for expression of, e.g, inducible or constitutive expression of a fusion protein comprising a translocation domain of the invention and a nucleic acid sequence amplified using a primer of the invention.

As used herein, a “stable cell line” refers to a cell line, which stably, i.e. over a prolonged period, expresses a heterologous nucleic sequence, i.e. a T1R or G protein. 20 In preferred embodiments, such stable cell lines will be produced by transfecting appropriate cells, typically mammalian cells, e.g. HEK-293 cells, with a linearized vector that contains a T1R expression construct, i.e. T1R1, T1R2 and/or T1R3. Most preferably, such stable cell lines will be produced by co-transfecting two linearized plasmids that express hTIRl and hTlR3 or hTlR2 and hTlR3 and an appropriate 25 selection procedure to generate cell lines having these genes stably integrated therein. Most preferably, the cell line will also stably express a G protein such as Goi5.

The phrase “selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture 30 (e.g., total cellular or library DNA or RNA).

The phrase “stringent hybridization conditions” refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but to no other sequences. Stringent conditions are sequence dependent 86 and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology -Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993). Generally, stringent conditions are selected to be about 5-10° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at-least about 30° C for short probes (e.g, 10 to 50 nucleotides) and at least about 60° C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1 % SDS at 65°C. Such hybridizations and wash steps can be carried out for, e.g, 1, 2, 5, 10, 15, 30, 60; or more minutes.

Nucleic acids that do not hybridize to each other under stringent conditions are still substantially related if the polypeptides that they encode are substantially related. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions. Exemplary “moderately stringent hybridization conditions” include a hybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37°C, and a wash in IX SSC at 45°C. Such hybridizations and wash steps can be carried out for, e.g, 1, 2, 5, 10, 15, 30, 60, or more minutes. A positive hybridization is at least twice background, Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. 87

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“Antibody” refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad 5 immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair 10 having one “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms "variable light chain" (VL) and "variable heavy chain" (VH) refer to these light and heavy chains respectively. 15 A “chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.', or 20 (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

An “anti-TIR” antibody is an antibody or antibody fragment that specifically binds a polypeptide encoded by a T1R gene, cDNA, or a subsequence thereof

The term “immunoassay” is an assay that uses an antibody to specifically bind 25 an antigen. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the antigen.

The phrase “specifically (or selectively) binds” or “specifically (or selectively) reacts with,” when referring to a molecule or composition, refers to a binding reaction that is determinative ofthe presence ofthe molecule in a heterogeneous population of 30 other biologies. Thus, under designated conditions, the specified molecules bind to a particular receptor at least two times the background and do not substantially bind in a significant amount to other molecules present in the sample. Specific binding to a 88

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receptor under such conditions may require a receptor that is selected for its specificity for a particular molecule.

Regarding antibodies, a variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid- 5 phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g, Harlow &amp;amp; Lane, Antibodies, A Laboratory Manual, (1988), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically a specific or selective reaction will be at least twice background signal or noise and more typically more than 10 to 10 100 times background.

The phrase “selectively associates with” refers to the ability of a nucleic acid to “selectively hybridize” with another as defined above, or the ability of an antibody to “selectively (or specifically) bind to a protein, as defined above.

The term “expression vector” refers to any recombinant expression system for 15 the purpose of expressing a nucleic acid sequence of the invention in vitro or in vivo, constitutively or inducibly, in any cell, including prokaryotic, yeast, fungal, plant, insect or mammalian cell. The term includes linear or circular expression systems. The term includes expression systems that remain episomal or integrate into the host cell genome. The expression systems can have the ability to self-replicate or not, i.e., drive 20 only transient expression in a cell. The term includes recombinant expression “cassettes which contain only the minimum elements needed for transcription of the recombinant nucleic acid.

By “host cell” is meant a cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells may be prokaryotic 25 cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, worm or mammalian cells such as CHO, Hela, HEK-293, and the like, e.g, cultured cells, explants, and cells in vivo.

Compounds

As discussed above, there are different domains on the T1R receptors. T1R1, 30 T1R2, and T1R3 each contain an N-terminal extracellular domain (also known as the

Venus flytrap domain), transmembrane domains comprising seven transmembrane regions, and corresponding cytoplasmic, and extracellular loops; a cysteine rich 89 domain, and a C-terminal domain. Each region defines a specific set of compounds that bind specifically to that region.

In humans, the N-terminal extracellular domain comprises amino acids 1 to 560 of hTlR2 and amino acids 1 to 563 ofhTlR3. In rats, the N-terminal extracellular domain comprises amino acids 1 to 564 of rTlR2, and amino acids 1 to 568 of rTlR3,

In humans, the C-terminal transmembrane domain and intracellular domain comprise amino acids 561 to 839 of hTlR2, and amino acids 564 to 852 of hTlR3. In rats, the C-terminal transmembrane domain and intracellular domain comprise amino acids 565 to 842 of rTlR2, and amino acids 569 to 858 of rTlR3.

Metabotropic glutamate receptors (mGluR) are another class of C-class G protein-coupled receptors that respond to glutamate. These are found primarily in the brain and neuronal tissue where they play a role in neuronal signaling. The mGluR N-terminal extracellular domain can be covalently linked to a T1R in order to create chimeric receptors. The mGluR receptor can be any of mGluRl-mGluR8, for example. Different ligands bind to different domains on different subunits of both the umami and the sweet receptors. For example, aspartame and neotame bind to the N-terminal extracellular domain of T1R2, while cyclamate, neohesperidin dihydrochalcone (NHDC), and lactisole bind to the transmembrane domain of T1R3, Because T1R3 is one of the two subunits in the T1R1/T1R3 umami taste receptor, cyclamate, NHDC and lactisole can interact with T1R3 in the T1R1/T1R3 umami taste receptor as well. Cyclamate and NHDC enhance the activity of the umami taste receptor, while lactisole inhibits the umami receptor.

The specific binding compounds of the invention as it relates to umami tastants comprise amides. The amide compounds also comprise certain sub-classes of amide derivatives or classes of derivatives related to amides, such as for example ureas, urethanes, oxalamides, acrylamides, and the like.

Molecules that interact with the transmembrane domain of T1R2, for example, can be modulators of sweet taste, and molecules that interact with the transmembrane domain of T1R3 can be modulators of sweet taste and/or umami taste.

Human T1R2/T1R3 recognizes a group of sweeteners which are not recognized by rat T1R2/T1R3, and human but not rat T1R2/T1R3 is inhibited by lactisole. When the extracellular domain of human T1R2 was replaced by its rat counterpart, the human receptor lost the ability to recognize aspartame, indicating that this part of human T1R2 90

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is required for binding to aspartame. Inversely, when the extracellular domain of rat T1R2 was replaced by its human counterpart, the rat receptor acquired the ability to recognize aspartame, indicating that this part of the human T1R2 is sufficient to bind aspartame. By the same principle, the transmembrane domain of human T1R3 was 5 required and sufficient for

Table 6 shows the abbreviations used to represent various rat/human chimeric receptors and receptor subunits. TABLE 6 hTlR2 - human T1R2 hTlR3 - human T1R3 rTlR2 - rat T1R2 rTlR3-rat T1R3 hTlR2/rTlR3 - a receptor composed of human T1R2 and rat T1R3 rTlR2/hTlR3 - a receptor composed of a rat T1R2 and human T1R3 hTlR2/h3-r3 - a receptor composed of human T1R2 and a chimeric T1R3 with human N-terminal extracellular domain and rat transmembrane and C-terminal domain rTlR2/r3-h3 - a receptor composed of rat T1R2 and a chimeric T1R3 with rat N-terminal extracellular domain and human transmembrane and C-terminal domain h2-r2/rTlR3 - a receptor composed of a chimeric T1R2 with human N-terminal extracellular domain and rat transmembrane and C-terminal domain and rat T1R3 r2-h2/rT!R3 - a receptor composed of a chimeric T1R2 with rat N-terminal extracellular domain and human transmembrane and C-terminal domain and rat T1R3 h2-hl/hTlR3 - a receptor composed of a chimeric T1R with human T1R2 N-terminal extracellular domain and human T1R1 transmembrane and C-terminal domain and human T1R3 hl-h2/hTlR3 - a receptor composed of a chimeric T1R with human T1R1 N-terminal extracellular domain and human T1R2 transmembrane and C-terminal domain and human T1R3 h2-mGluRl/h3-mGluRl - a receptor composed of a N-terminal extracellular domain from hTlR2 covalently linked to the transmembrane and C-terminal domain of mGluRl and a N-terminal extracellular domain from hTlR3 covalently linked to the transmembrane and C-terminal domain of mGluRl hl-mGlulR/h3-mGluRl - a receptor composed of a N-terminal extracellular domain from hTIRl covalently linked to the transmembrane and C-terminal domain of mGluRland a N-terminal extracellular domain from hTlR3 covalently linked to the transmembrane and C-terminal domain of mGluRl mGluRl-h2/mGluRl-h3 - a receptor composed of a N-terminal extracellular domain from mGluRl covalently linked to the transmembrane and C-terminal domain of hTlR2 and a N-terminal extracellular domain from a mGluRl covalently linked to the transmembrane and C-terminal domain of hTlR3 mGluRl-hl/mGluRl-h3 - receptor composed of a N-terminal extracellular domain from mGluRl covalently linked to the transmembrane and C-terminal domair\of hTIRl and a N-terminal extracellular domain from mGluRl covalently linked to the transmembrane and C-terminal domain of hTlR3 91

Disclosed herein are non-naturally occurring compounds that specifically bind to the T1R2/T1R3 receptor comprising hTlR2/hTlR3 but not rTlR2/rTlR3. Examples of such compounds include, but are not limited to neotame, aspartame, cyclamate, lactisol, Compound 883360, Compound 6542888, Compound 403249, Compound 6364395, Dihydroxybenzoic acid (DHB), Compound 6542888, and neohesperidine dihydrochalcone (NHDC) Additional examples are found in Tables 1-4. The organic, non-peptide compounds can be approximately the size of a box of dimensions 15x8x8 angstroms, more preferably the dimension should be 12x5x5 angstroms.

Also disclosed are compounds that specifically bind to a T1R2/T1R3 receptor comprising hTlR2/rTlR3 but not rTlR2/hTlR3. Examples of such compounds include, but are not limited to aspartame, and neotame. Additional examples are found in Table 5.

Also disclosed are compounds that specifically bind to the N-terminal extracellular domain of T1R2 of the hTlR2/hTlR3 receptor. Examples of such compounds include, but are not limited to neotame, aspartame carbohydrate sugars (e.g. sucrose, fructose, glucose, tagatose, erythritol, sorbitol, maltose, xylitol, lactose and galactose, as well as all other carbohydrate sugars). Additional examples are found in Tables.

Also disclosed are compounds that specifically bind to the Venus Flytrap Domain (VFD) of T1R2 of the hTlR2/hTlR3 and hTlR2/rTlR3 receptor.

Also disclosed are compounds that specifically bind to the N-terminal Venus flytrap domain of the T1R2 subunit of the T1R2/T1R3 receptor. More specifically, also disclosed are compounds that specifically bind to amino acid residues 144 and 302 of the human N-terminal Venus flytrap domain of the T1R2 subunit of theTlR2/TlR3 receptor. Examples of such compounds include, but are not limited to aspartame, neotame, carbohydrates, and sweet amino acids, such as D-Trp, Ala, and Gly.

Also disclosed are compounds that specifically bind to the cysteine-rich region of T1R2 of the hTlR2/hTlR3 receptor. Also disclosed are compounds that specifically bind to the Transmembrane Domain (TM) of T1R2 of the hTlR2/hTlR3 receptor.

Also disclosed are compounds that specifically bind to a T1R2/T1R3 receptor comprising rTlR2/hTlR3 but not hTlR2/rTlR3. Examples of such compounds include, but are not limited to cyclamate, NHDC, lactisole, Compound 883360, 92

Compound 403249, and Compound 6364395. Additional examples are found in Table 5.

Also disclosed are compounds that specifically bind to hTlR2/hTlR3 and rTlR2/r3-h3 but not to rTlR2/rTlR3 or to hTlR2/h3-r3. Examples of such compounds include, but are not limited to cyclamate, NHDC, lactisole, Compound 883360, Compound 403249 and Compound 6364395,

Also disclosed are compounds that specifically bind to extracellular loop 2 and extracellular loop 3 of the human C-terminal domain of the T1R3 subunit of theTlR2/TlR3 receptor. Also disclosed are compounds that specifically bind to hTlR2/hTlR3 and r2-h2/rTlR3 but not to rTlR2/rTlR3 or to h2-r2/hTlR3.

Also disclosed are compounds that specifically bind to the human N-terminal extracellular domain of the T1R3 subunit of the T1R2/T1R3 receptor. Also disclosed are compounds that specifically bind to the Venus Flytrap Domain (VFD) of T1R3 of the hTlR2/hTlR3 receptor. Examples of such compounds include, but are not limited to aspartame, neotame, carbohydrates, and sweet amino acids, such as D-Trp, Ala, and Gly.

Also disclosed are compounds that specifically bind to the Transmembrane Domain of T1R3 of the hTlR2/hTlR3 receptor. Also disclosed are compounds that specifically bind to extracellular loop 2 and extracellular loop 3 of the.human transmembrane domain of the T1R3 subunit of T1R2/T1R3. Examples of such compounds include, but are not limited to cyclamate.

The compound of the invention does not include sucrose, fructose, glucose, erythritol, isomalt, lactitol, mannitol, sorbitol, xylitol, certain known natural terpenoids, flavonoids, or protein sweeteners, di-peptides, tri-peptides, aspartame, saccharin, sucralose, halogenated saccharides, acesulfame-K, cyclamate, sucralose, and alitame. neotame, perillartine, SC-45647, SC-40014, monellin, NC-002740-01, thaumatin, CC-00100, NC-00420, alitame, SC-44102, dulcin, NC-00576, slycyrrhizic Acid, stevioside, Na-Saccharin, D-tryptophan, cyclamate, DHB, glycolic Acid, glycine, D (-)fructose, homofuronol, D (-) tagatose, maltose, D (+) glucose, D-sorbitol, D (+) galactose, a-lactose, L()fructose, L (+) Compound 403249, and glucose,

Optionally, a compound of the invention is also not Compound 6364395.

Also disclosed herein are compounds that bind a truncated region of a T1R domain. For example, disclosed are compounds that specifically bind to the TM domain 93 of T1R2 of a truncated sweet receptor comprising h2TM/h3TM, compounds that specifically bind to the TM domain of T1R3 of a truncated sweet receptor comprising h2TM/h3TM, compounds that specifically bind to the TM domain of T1R2 of a chimeric receptor comprising mGluR-h2/mGluR-h3, compounds that specifically bind to the TM domain of T1R3 of a chimeric receptor comprising mGluR-h2/mGluR-h3, compounds that binds to the TM domain of T1R1 of a truncated savory receptor comprising hlTM/h3TM, compound that binds to the TM domain of T1R3 of a truncated sweet receptor comprising hlTM/h3TM, compounds that bind to the TM domain of T1R1 of a chimeric receptor comprising mGluR-hl/mGiuR-h3, and compounds that bind to the TM domain of T1R3 of a chimeric receptor comprising mGluR-hl/mGluR-h3. SEQ ID NOS: 29-33 represent these truncated receptors.

The compounds ofthe invention do not include monosodium glutamate (“MSG”), inosine monophosphate (IMP) or guanosine monophosphate (GMP), sucrose, fructose, glucose, erythritol, isomalt, lactitol, mannitol, sorbitol, xylitol, certain known natural terpenoids, flavonoids, or protein sweeteners, di-peptides, tri-peptides aspartame, saccharin, sucralose, halogenated saccharides, acesulfame-K, cyclamate, sucralose, alitame, monosodium glutamate (“MSG”), inosine monophosphate (IMP) or guanosine monophosphate (GMP), or adenosine monophospate.

Compound 403249 is (5-(4H-benzo[d][l,3]oxathiin-2-yl)-2-methyoxyphenol, while Compound 6364395 is 3-(3-hydroxy-4-methoxyphenethyl)benzo[d]isoxyazoIe- 4,6-diol.

The compounds described above can demonstrate a compound-dependent increase in fluorescence with an activity compared to the maximal activity for fructose of at least 25% in a fluorescence-based assay using a FLIPR instrument (Fluorometric Intensity Plate Reader, Molecular Devices, Sunnyvale, CA). For examples of this protocol, see Examples 12 and 18. The compounds can also demonstrate a compound-dependent decrease in the ECSO for a sweetener by at least two-fold in a fluorescence-based assay using a FLIPR (Molecular Devices) instrument. Furthermore, in a cell-based assay, the compound can result in at least 10 out of 100 cells transfected with wild-type or chimeric receptor showing a compound-dependent increase in fluorescence. An example of a cell-based assay can be found in Example 24. The compound can also demonstrate a compound-dependent increase of at least 1.1, 1.2, 1.3, 1.4, 1.5, 1,6, 1.7, 1.8, 1.9, two-fold or greater, or any number in between, in the 94

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number of fluorescent cells in response to a sub-maximal level of a sweetener. The response can be measured by fluorescence, calcium levels, IP3 levels, cAMP levels, GTPyS binding, or reporter gene activity (e.g. luciferase, beta-galactosidase).

Furthermore, the compounds disclosed herein can have one or more of the 5 following characteristics in a cell: a decreased EC50 compared to a control of at least approximately 50%, increased intracellular Ca2 level by at least approximately 25%, increased intracellular cAMP by at least approximately 25%, increased intracellular cGMP by at least approximately 25%, increased intracellular IP3 by at least approximately 25%, or increased G protein binding of GTPyS by at least approximately 10 25%.

Methods of Using the Compounds

Also disclosed are methods modulating the savory taste of a comestible or medicinal product comprising providing at least one comestible or medicinal product, 15 or a precursor thereof, and combining the comestible or medicinal product or precursor thereof with at least a savory flavor modulating amount of at least one non-naturally occurring compound as disclosed herein, or a comestibly acceptable salt thereof, so as to form a modified comestible or medicinal product; thereby modulating the savory taste of a comestible or medicinal product. 20 Also disclosed are methods for inhibiting the savory taste of a comestible or medicinal product comprising providing at least one comestible or medicinal product, or a precursor thereof, and combining the comestible or medicinal product or precursor thereof with at least a savory flavor inhibiting amount of at least one non-naturally occurring compound as disclosed herein, or a comestibly acceptable salt thereof, so as 25 to form a modified comestible or medicinal product; thereby inhibiting the savory taste of a comestible or medicinal product.

Also disclosed are methods for increasing the savory taste of a comestible or medicinal product comprising providing at least one comestible or medicinal product, or a precursor thereof, and combining the comestible or medicinal product or precursor 30 thereof with at least a savory flavor increasing amount of at least one non-naturally occurring compound as disclosed herein, or a comestibly acceptable salt thereof, so as to form a modified comestible or medicinal product; thereby increasing the savory taste of a comestible or medicinal product. 95

Also disclosed are methods for modulating the sweet taste of a comestible or medicinal product comprising providing at least one comestible or medicinal product, or a precursor thereof, and combining the comestible or medicinal product or precursor thereof with at least a sweet flavor modulating amount of at least one non-naturally occurring compound as disclosed herein, or a comestibly acceptable salt thereof, so as to form a modified comestible or medicinal product; thereby modulating the sweet taste of a comestible or medicinal product.

Also disclosed are methods for inhibiting the sweet taste of a comestible or medicinal product comprising providing at least one comestible or medicinal product, or a precursor thereof, and combining the comestible or medicinal product or precursor thereof with at least a sweet flavor inhibiting amount of at least one non-naturally occurring compound as disclosed herein, or a comestibly acceptable salt thereof, so as to form a modified comestible or medicinal product; thereby inhibiting the sweet taste of a comestible or medicinal product.

Also disclosed are methods for increasing the sweet taste of a comestible or medicinal product comprising providing at least one comestible or medicinal product, or a precursor thereof, and combining the comestible or medicinal product or precursor thereof with at least a sweet flavor increasing amount of at least one non-naturally occurring compound as disclosed herein, or a comestibly acceptable salt thereof, so as to form a modified comestible or medicinal product; thereby increasing the sweet taste of a comestible or medicinal product.

Also disclosed are methods of enhancing umami taste perception comprising contacting an umami receptor with cyclamate and NHDC, and their derivatives, as well as methods of enhancing umami taste perception comprising contacting an umami receptor with lactisole derivatives. Also disclosed are methods of enhancing sweet taste perception comprising contacting an sweet receptor with cyclamate and NHDC, and their derivatives. Also disclosed are methods of enhancing sweet taste perception comprising contacting an sweet receptor with lactisole derivatives.

Isolation and Expression ofTIR Polypeptides

Isolation and expression of the TIRs, or fragments or variants thereof, of the invention can be performed as described below. PCR primers can be used for the 96 amplification of nucleic acids encoding taste receptor ligand-binding regions, and libraries of these nucleic acids can optionally be generated. Individual expression vectors or libraries of expression vectors can then be used to infect or transfect host cells for the functional expression of these nucleic acids or libraries. These genes and vectors can be made and expressed in vitro or in vivo. One of skill will recognize that desired phenotypes for altering and controlling nucleic acid expression can be obtained by modulating the expression or activity of the genes and nucleic acids (e.g, promoters, enhancers and the like) within the vectors of the invention. Any of the known methods described for increasing or decreasing expression or activity can be used. The invention can be practiced in conjunction with any method or protocol known in the art, which are well described in the scientific and patent literature.

The nucleic acid sequences of the invention and other nucleic acids used to practice this invention, whether RNA, cDNA, genomic DNA, vectors, viruses or hybrids thereof, may be isolated from a variety of sources, genetically engineered, amplified, and/or expressed recombinantly. Any recombinant expression system can be used, including, in addition to mammalian cells, e.g, bacterial, yeast, insect, or plant systems.

Alternatively, these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g, Carruthers, Cold Spring Harbor Symp. Quant. Biol. 47:411-418 (1982); Adams, Ant Chem. Soc. 105:661 (1983); Belousov, Nucleic Acids Res. 25:3440-3444 (1997); Frenkel, Free Radic. Biol. Med. 19:373-380 (1995); Blommers, Biochemistry 33:7886-7896 (1994); Narang, Meth. Enzymol. 68:90 (1979); Brown, Meth. Enzymol. 68:109 (1979); Beaucage, Tetra. Lett. 22:1859 (1981); U.S. Patent No. 4,458,066. Double-stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.

Techniques for the manipulation of nucleic acids, such as, for example, for generating mutations in sequences, subcloning, labeling probes, sequencing, hybridization and the like are well described in the scientific and patent literature. See, e.g, Sambrook, ed., Molecular Cloning: a Laboratory manual (2nd ed.), Vols. 1-3, Cold Spring Harbor Laboratory (1989); Current Protocols in Molecular Biology, Ausubel, ed. John Wiley &amp;amp; Sons, Inc., New York (1997); Laboratory Techniques in 97

Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, Part I, Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Nucleic acids, vectors, capsids, polypeptides, and the like can be analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, e.g., analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, e.g, fluid or gel precipitin reactions, immunodiffusion, immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immuno-fluorescent assays, Southern analysis, Northern analysis, dot-blot analysis, gel electrophoresis (e.g., SDS-PAGE), RT-PCR, quantitative PCR, other nucleic acid or target or signal amplification methods, radiolabeling, scintillation counting, and affinity chromatography.

Oligonucleotide primers may be used to amplify nucleic acid fragments encoding taste receptor ligand-binding regions. The nucleic acids described herein can also be cloned or measured quantitatively using amplification techniques.

Amplification methods are also well known in the art, and include, e.g., polymerase chain reaction, PCR (PCR Protocols, a Guide to Methods and Applications, ed. Innis. Academic Press, N.Y. (1990) and PCR Strategies, ed. Innis, Academic Press, Inc., N.Y. (1995), ligase chain reaction (LCR) (see, e.g., Wu, Genomics 4:560 (1989); Landegren, Science 241:1077, (1988); Barringer, Gene 89:117 (1990)); transcription amplification (see, e.g, Kwoh, Proc. Natl. Acad Sci. USA 86:1173 (1989)); and, self-sustained sequence replication (see, e.g, Guatelli, Proc. Natl. Acad. Sci. USA 87:1874 (1990)); Q Beta replicase amplification (see, e.g., Smith, J. Clin. Microbiol. 35:1477-1491 (1997)); automated Q-beta replicase amplification assay (see, e.g., Burg, Mol. Cell. Probes 10:257-271 (1996)) and other RNA polymerase mediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); see also Berger, Methods Enzymol. 152:307-316 (1987); Sambrook; Ausubel; U.S. Patent Nos. 4,683,195 and 4,683,202; Sooknanan, Biotechnology 13:563-564 (1995). The primers can be designed to retain the original sequence of the “donor” 7-membrane receptor. Alternatively, the primers can encode amino acid residues that are conservative substitutions (e.g., hydrophobic for hydrophobic residue, see above discussion) or functionally benign substitutions (e.g, do not prevent plasma membrane insertion, cause cleavage by peptidase, cause 98

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abnormal folding of receptor, and the like). Once amplified, the nucleic acids, either individually or as libraries, may be cloned according to methods known in the art, if desired, into any of a variety of vectors using routine molecular biological methods; methods for cloning in vitro amplified nucleic acids are described, e.g., U.S. Pat. No, 5 5,426,039.

The primer pairs may be designed to selectively amplify ligand-binding regions of the T1R family members. These regions may vary for different ligands or tastants. Thus, what may be a minimal binding region for one tastant, may be too limiting for a second tastant. Accordingly, ligand-binding regions of different sizes comprising 10 different extracellular domain structures may be amplified.

Paradigms to design degenerate primer pairs are well known in the art. For example, a COnsensus-DEgenerate Hybrid Oligonucleotide Primer (CODEHOP) strategy computer program is accessible as http://blocks.fhcrc.org/codehop.html, and is directly linked from the BlockMaker multiple sequence alignment site for hybrid 15 primer prediction beginning with a set of related protein sequences, as known taste receptor ligand-binding regions (see, e.g, Rose, Nucleic Acids Res. 26:1628-1635 (1998); Singh, Biotechniques 24:318-319 (1998)).

Means to synthesize oligonucleotide primer pairs are well known in the art. “Natural” base pairs or synthetic base pairs can be used. For example, use of artificial 20 nucleobases offers a versatile approach to manipulate primer sequence and generate a more complex mixture of amplification products. Various families of artificial nucleobases are capable of assuming multiple hydrogen bonding orientations through internal bond rotations to provide a means for degenerate molecular recognition. Incorporation of these analogs into a single position of a PCR primer allows for 25 generation of a complex library of amplification products. See, e.g., Hoops, Nucleic Acids Res. 25:4866-4871 (1997). Nonpolar molecules can also be used to mimic the shape of natural DNA bases. A non-hydrogen-bonding shape mimic for adenine can replicate efficiently and selectively against a nonpolar shape mimic for thymine (see, e.g, Morales, Nat. Struct. Biol. 5:950-954 (1998)). For example, two degenerate bases 30 can be the pyrimidine base 6H, 8H-3,4-dihydropyrimido[4,5-c][l,2]oxazin-7-one or the purine base N6-methoxy-2,6-diaminopurine (see, e.g, Hill, Proc. Natl. Acad Sci. USA 95:4258-4263 (1998)). Exemplary degenerate primers of the invention incorporate the nucleobase analog 5 ’-Dimethoxytrityl-N-benzoyl-2’-deoxy-Cytidine,3 ’-[(2- 99

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cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite (the term “P” in the sequences, see above). This pyrimidine analog hydrogen bonds with purines, including A and G residues.

Polymorphic variants, alleles, and interspecies homologs that are substantially

5 identical to a taste receptor disclosed herein can be isolated using the nucleic acid probes described above. Alternatively, expression libraries can be used to clone T1R polypeptides and polymorphic variants, alleles, and interspecies homologs thereof, by detecting expressed homologs immunologically with antisera or purified antibodies made against a T1R polypeptide, which also recognize and selectively bind to the T1R 10 homolog.

Nucleic acids that encode ligand-binding regions of taste receptors may be generated by amplification (e.g, PCR) of appropriate nucleic acid sequences using degenerate primer pairs. The amplified nucleic acid can be genomic DNA from any cell or tissue or mRNA or cDNA derived from taste receptor-expressing cells. 15 In one embodiment, hybrid protein-coding sequences comprising nucleic acids encoding TIRs fused to translocation sequences may be constructed. Also provided are hybrid TIRs comprising the translocation motifs and tastant-binding domains of other families of chemosensory receptors, particularly taste receptors. These nucleic acid sequences can be operably linked to transcriptional or translational control elements, 20 e.g, transcription and translation initiation sequences, promoters and enhancers, transcription and translation terminators, polyadenylation sequences, and other sequences useful for transcribing DNA into RNA. In constitutive of recombinant expression cassettes, vectors, and transgenics, a promoter fragment can be employed to direct expression of the desired nucleic acid in all desired cells or tissues. 25 In another embodiment, fusion proteins may include C-terminal or N-terminal translocation sequences. Further, fusion proteins can comprise additional elements, e.g, for protein detection, purification, or other applications. Detection and purification facilitating domains include, e.g, metal chelating peptides such as polyhistidine tracts, histidine-tryptophan modules, or other domains that allow 30 purification on immobilized metals; maltose binding protein; protein A domains that allow purification on immobilized immunoglobulin; or the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp., Seattle, WA). 100

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The inclusion of a cleavable linker sequences such as Factor Xa (see, e.g.,

Ottavi, Biochimie 80:289-293 (1998)), subtilisin protease recognition motif (see, e.g, Polyak, Protein Eng. 10:615-619 (1997)); enterokinase (Invitrogen, San Diego, CA), and the like, between the translocation domain (for efficient plasma membrane 5 expression) and the rest of the newly translated polypeptide may be useful to facilitate purification. For example, one construct can include a polypeptide encoding a nucleic acid sequence linked to six histidine residues followed by a thioredoxin, an enterokinase cleavage site (see, e.g, Williams, Biochemistry 34:1787-1797 (1995)), and an C-terminal translocation domain. The histidine residues facilitate detection and 10 purification while the enterokinase cleavage site provides a means for purifying the desired protein(s) from the remainder of the fusion protein. Technology pertaining to vectors encoding fusion proteins and application of fusion proteins are well described in the scientific and patent literature, see, e.g, Kroll, DNA Cell. Biol. 12:441-53 (1993).

Expression vectors, either as individual expression vectors or as libraries of 15 expression vectors, comprising the ligand-binding domain encoding sequences may be introduced into a genome or into the cytoplasm or a nucleus of a cell and expressed by a variety of conventional techniques, well described in the scientific and patent literature. See, e.g, Roberts, Nature 328:731 (1987); Berger supra, Schneider, Protein Expr. Purif. 6435:10 (1995); Sambrook; Tijssen; Ausubel. Product information from 20 manufacturers of biological reagents and experimental equipment also provide information regarding known biological methods. The vectors can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries, or prepared by synthetic or recombinant methods.

The nucleic acids can be expressed using expression cassettes, vectors or 25 viruses which are stably or transiently expressed in cells (e.g, episomal expression systems). Selection markers can be incorporated into expression cassettes and vectors to confer a selectable phenotype on transformed cells and sequences. For example, selection markers can code for episomal maintenance and replication such that integration into the host genome is not required. For example, the marker may encode 30 antibiotic resistance (e.g, chloramphenicol, kanamycin, G418, blasticidin, hygromycin) or herbicide resistance (e.g, chlorosulfuron or Basta) to permit selection of those cells transformed with the desired DNA sequences (see, e.g, Blondelet-Rouault, Gene 190:315-317 (1997); Aubrecht, J. Pharmacol. Exp. Ther. 281:992-997 (1997)). 101

Because selectable marker genes conferring resistance to substrates like neomycin or hygromycin can only be utilized in tissue culture, chemoresistance genes are also used as selectable markers in vitro and in vivo. A chimeric nucleic acid sequence may encode a T1R ligand-binding domain within any 7-transmembrane polypeptide. Because 7-transmembrane receptor polypeptides have similar primary sequences and secondary and tertiary structures, structural domains (e.g., extracellular domain, TM domains, cytoplasmic domain, etc.) can be readily identified by sequence analysis. For example, homology modeling, Fourier analysis and helical periodicity detection can identify and characterize the seven domains with a 7-transmembrane receptor sequence. Fast Fourier Transform (FFT) algorithms can be used to assess the dominant periods that characterize profiles of the hydrophobicity and variability of analyzed sequences. Periodicity detection enhancement and alpha helical periodicity index can be done as by, e.g, Donnelly, Protein Sci. 2:55-70 (1993). Other alignment and modeling algorithms are well known in the art, see, e.g, Peitsch, Receptors Channels 4:161-164 (1996); Kyte &amp;amp; Doolittle, J. Med. Bio., 157:105-132 (1982); Cronet, Protein Eng. 6:59-64 (1993).

The present invention also includes not only the DNA and proteins having the specified nucleic and amino acid sequences, but also DNA fragments, particularly fragments of, e.g., 40, 60, 80, 100, 150, 200, or 250 nucleotides, or more, as well as protein fragments of, e.g., 10, 20, 30, 50, 70, 100, or 150 amino acids, or more. Optionally, the nucleic acid fragments can encode an antigenic polypeptide, which is capable of binding to an antibody raised against a T1R family member. Further, a protein fragment of the invention can optionally be an antigenic fragment, which is capable of binding to an antibody raised against a T1R family member.

Also contemplated are chimeric proteins, comprising at least 10, 20, 30, 50, 70, 100, or 150 amino acids, or more, of one of at least one of the T1R polypeptides described herein, coupled to additional amino acids representing all or part of another GPCR, preferably a member ofthe 7 transmembrane superfamily. These chimeras can be made from the instant receptors and another GPCR, or they can be made by combining two or more of the present T1R receptors. In one embodiment, one portion of the chimera corresponds to or is derived from the extracellular domain of a T1R polypeptide of the invention. In another embodiment, one portion of the chimera corresponds to, or is derived from the extracellular domain and one or more of the 102

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transmembrane domains of a T1R polypeptide described herein, and the remaining portion or portions can come from another GPCR. Chimeric receptors are well known in the art, and the techniques for creating them and the selection and boundaries of domains or fragments of G protein-coupled receptors for incorporation therein are also 5 well known. Thus, this knowledge of those skilled in the art can readily be used to create such chimeric receptors. The use of such chimeric receptors can provide, for example, a taste selectivity characteristic of one of the receptors specifically disclosed herein, coupled with the signal transduction characteristics of another receptor, such as a well known receptor used in prior art assay systems. 10 As noted above, such chimeras, analogous to the native T1R receptor, or native T1R receptor combination or association will bind to and/or be activated by molecules that normally affect sweet taste or umami taste. Functional chimeric T1R receptors or receptor combinations are molecules which when expressed alone or in combination with other TIRs or other GPCRs (which may themselves be chimeric) bind to or which 15 are activated by taste stimuli, particularly sweet (T1R2/3) or umami taste stimuli (T1R1/3). Molecules that elicit sweet taste include natural and artificial sweeteners such as sucrose, aspartame, xylitol, cyclamate, et al., Molecules that elicit umami taste include glutamate and glutamate analogs and other compounds that bind to native T1R1 and/or T1R3, such as 5’-nucleotides. 20 For example, a domain such as a ligand-binding domain, an extracellular domain, a transmembrane domain, a transmembrane domain, a cytoplasmic domain, an N-terminal domain, a C-terminal domain, or any combination thereof, can be covalently linked to a heterologous protein. For instance, an T1R extracellular domain can be linked to a heterologous GPCR transmembrane domain, or a heterologous 25 GPCR extracellular domain can be linked to a T1R transmembrane domain. Other heterologous proteins of choice can be used; e.g., green fluorescent protein.

Also within the scope of the invention are host cells for expressing the TIRs, fragments, chimeras or variants of the invention. To obtain high levels of expression of a cloned gene or nucleic acid, such as cDNAs encoding the TIRs, fragments, or 30 variants of the invention, one of skill typically subclones the nucleic acid sequence of interest into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator, and if for a nucleic acid encoding a protein, a ribosome binding site for translational initiation. Suitable bacterial promoters are well 103 known in the art and described, e.g., in Sambrook et al. However, bacterial or eukaryotic expression systems can be used,

Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g, Sambrook et al.) It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at lest one nucleic acid molecule into the host cell capable of expressing the T1R, fragment, or variant of interest.

After the expression vector is introduced into the cells, the transfected cells are cultured under conditions favoring expression of the receptor, fragment, or variant of interest, which is then recovered from the culture using standard techniques. Examples of such techniques are well known in the art. See, e.g, WO 00/06593, which is incorporated by reference in a manner consistent with this disclosure.

Detection ofTIR polypeptides

In addition to the detection ofTIR genes and gene expression using nucleic acid hybridization technology, one can also use immunoassays to detect TIRs, e.g, to identify taste receptor cells, and variants ofTIR family members. Immunoassays can be used to qualitatively or quantitatively analyze the TIRs. A general overview of the applicable technology can be found in Harlow &amp;amp; Lane, Antibodies: A Laboratory Manual (1988). 1. Antibodies to T1R family members

Methods of producing polyclonal and monoclonal antibodies that react specifically with a T1R family member are known to those of skill in the art (see, e.g, Coligan, Current Protocols in Immunology (1991); Harlow &amp;amp; Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice (2d ed. 1986); and Kohler &amp;amp; Milstein, Nature, 256:495-497 (1975)). Such techniques include antibody preparation by selection of antibodies from libraries of recombinant antibodies in phage or similar vectors, as well as preparation of polyclonal and monoclonal antibodies by immunizing 104 rabbits or mice (see, e.g., Huse et ai, Science, 246:1275-1281 (1989); Ward et al., Nature, 341:544-546 (1989)). A number of TIR-comprising immunogens may be used to produce antibodies specifically reactive with a T1R family member. For example, a recombinant T1R polypeptide, or an antigenic fragment thereof, can be isolated as described herein. Suitable antigenic regions include, e.g., the consensus sequences that are used to identify members of the T1R family. Recombinant proteins can be expressed in eukaryotic or prokaryotic cells as described above, and purified as generally described above. Recombinant protein is the preferred immunogen for the production of monoclonal or polyclonal antibodies. Alternatively, a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen. Naturally occurring protein may also be used either in pure or impure form. The product is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies may be generated, for subsequent use in immunoassays to measure the protein.

Methods of production of polyclonal antibodies are known to those of skill in the art. For example, an inbred strain of mice (e.g., BALB/C mice) or rabbits is immunized with the protein using a standard adjuvant, such as Freund’s adjuvant, and a standard immunization protocol. The animal’s immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the T1R. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the protein can be done if desired (see Harlow &amp;amp; Lane, supra).

Monoclonal antibodies may be obtained by various techniques familiar to those skilled in the art. Briefly, spleen cells from an animal immunized with a desired antigen may be immortalized, commonly by fusion with a myeloma cell (see Kohler &amp;amp; Milstein, Eur. J. Immunol., 6:511-519 (1976)). Alternative methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enhanced by various techniques, including injection into the peritoneal cavity of a 105

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vertebrate host. Alternatively, one may isolate DNA sequences which encode a monoclonal antibody or a binding fragment thereof by screening a DNA library from human B cells according to the general protocol outlined by Huse et al., Science, 246:1275-1281 (1989). 5 Monoclonal antibodies and polyclonal sera are collected and titered against the immunogen protein in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Typically, polyclonal antisera with a titer of 104 or greater are selected and tested for their cross reactivity against non-TIR polypeptides, or even other T1R family members or other related proteins from other 10 organisms, using a competitive binding immunoassay. Specific polyclonal antisera and monoclonal antibodies will usually bind with a Kd of at least about 0.1 mM, more usually at least about 1 pM, optionally at least about 0.1 pM or better, and optionally 0.01 pM or better.

Once T1R family member specific antibodies are available, individual T1R 15 proteins and protein fragments can be detected by a variety of immunoassay methods. For a review of immunological and immunoassay procedures, see Basic and Clinical Immunology (Stites &amp;amp; Terr eds., 7th ed. 1991). Moreover, the immunoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in Enzyme Immunoassay (Maggio, ed., 1980); and Harlow &amp;amp; 20 Lane, supra. 2. Immunological binding assays T1R proteins, fragments, and variants can be detected and/or quantified using any of a number of well-recognized immunological binding assays (see, e.g, U.S. 25 Patents 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review ofthe general immunoassays, see also Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites &amp;amp; Terr, eds., 7th ed. 1991). Immunological binding assays (or immunoassays) typically use an antibody that specifically binds to a protein or antigen of choice (in this case a T1R family member 30 or an antigenic subsequence thereof). The antibody (e.g, anti-TIR) may be produced by any of a number of means well known to those of skill in the art and as described above. 106

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Immunoassays also often use a labeling agent to specifically bind to and label the complex formed by the antibody and antigen. The labeling agent may itself be one of the moieties comprising the antibody/antigen complex. Thus, the labeling agent may be a labeled T1R polypeptide or a labeled anti-TIR antibody. Alternatively, the 5 labeling agent may be a third moiety, such a secondary antibody that specifically binds to the antibody/TIR complex (a secondary antibody is typically specific to antibodies of the species from which the first antibody is derived). Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G may also be used as the label agent. These proteins exhibit a strong non-immunogenic 10 reactivity with immunoglobulin constant regions from a variety of species (see, e.g, Kronval et ai, J. Immunol., 111:1401-1406 (1973); Akerstrom et ai, J. Immunol., 135:2589-2542 (1985)). The labeling agent can be modified with a detectable moiety, such as biotin, to which another molecule can specifically bind, such as streptavidin. A variety of detectable moieties are well known to those skilled in the art. 15 Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, optionally from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, antigen, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient 20 temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C. A. Non-competitive assay formats

Immunoassays for detecting a T1R polypeptide in a sample may be either 25 competitive or noncompetitive. Noncompetitive immunoassays are assays in which the amount of antigen is directly measured. In one preferred “sandwich” assay, for example, the anti-TIR antibodies can be bound directly to a solid substrate on which they are immobilized. These immobilized antibodies then capture the T1R polypeptide present in the test sample. The T1R polypeptide is thus immobilized is then bound by a 30 labeling agent, such as a second T1R antibody bearing a label. Alternatively, the second antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second or third antibody is typically modified with a detectable moiety, 107 such as biotin, to which another molecule specifically binds, e.g., streptavidin, to provide a detectable moiety. B. Competitive assay formats

In competitive assays, the amount of T1R polypeptide present in the sample is measured indirectly by measuring the amount of a known, added (exogenous) T1R polypeptide displaced (competed away) from an anti-TIR antibody by the unknown T1R polypeptide present in a sample. In one competitive assay, a known amount of T1R polypeptide is added to a sample and the sample is then contacted with an antibody that specifically binds to the T1R. The amount of exogenous T1R polypeptide bound to the antibody is inversely proportional to the concentration of T1R polypeptide present in the sample. In a particularly preferred embodiment, the antibody is immobilized on a solid substrate. The amount of T1R polypeptide bound to the antibody may be determined either by measuring the amount of T1R polypeptide present in a TIR/antibody complex, or alternatively by measuring the amount of remaining uncomplexed protein. The amount of T1R polypeptide may be detected by providing a labeled T1R molecule. A hapten inhibition assay is another preferred competitive assay. In this assay the known T1R polypeptide is immobilized on a solid substrate. A known amount of anti-TIR antibody is added to the sample, and the sample is then contacted with the immobilized T1R. The amount of anti-TIR antibody bound to the known immobilized T1R polypeptide is inversely proportional to the amount of T1R polypeptide present in the sample. Again, the amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. Detection may be direct where the antibody is labeled or indirect by the subsequent addition of a labeled moiety that specifically binds to the antibody as described above. C. Cross-reactivity determinations

Immunoassays in the competitive binding format can also be used for crossreactivity determinations. For example, a protein at least partially encoded by the nucleic acid sequences disclosed herein can be immobilized to a solid support. Proteins (e.g, T1R polypeptides and homologs) are added to the assay that compete for binding 108 of the antisera to the immobilized antigen. The ability of the added proteins to compete for binding of the antisera to the immobilized protein is compared to the ability of the T1R polypeptide encoded by the nucleic acid sequences disclosed herein to compete with itself. The percent cross-reactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% cross-reactivity with each of the added proteins listed above are selected and pooled. The cross-reacting antibodies are optionally removed from the pooled antisera by immunoabsorption with the added considered proteins, e.g., distantly related homologs. In addition, peptides comprising amino acid sequences representing conserved motifs that are used to identify members ofthe T1R family can be used in cross-reactivity determinations.

The immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein, thought to be perhaps an allele or polymorphic variant of a T1R family member, to the immunogen protein (i.e., T1R polypeptide encoded by the nucleic acid sequences disclosed herein). In order to make this comparison, the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required to inhibit 50% of binding is less than 10 times the amount ofthe protein encoded by nucleic acid sequences disclosed herein required to inhibit 50% of binding, then the second protein is said to specifically bind to the polyclonal antibodies generated to a T1R immunogen.

Antibodies raised against T1R conserved motifs can also be used to prepare antibodies that specifically bind only to GPCRs of the T1R family, but not to GPCRs from other families.

Polyclonal antibodies that specifically bind to a particular member of the TI R family can be made by subtracting out cross-reactive antibodies using other T1R family members. Species-specific polyclonal antibodies can be made in a similar way. For example, antibodies specific to human T1R1 can be made by, subtracting out antibodies that are cross-reactive with orthologous sequences, e.g., rat T1R1 or mouse T1R1. D. Other assay formats

Western blot (immunoblot) analysis is used to detect and quantify the presence of T1R polypeptide in the sample. The technique generally comprises separating 109

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sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind the T1R polypeptide. The anti-TIR polypeptide antibodies 5 specifically bind to the T1R polypeptide on the solid support. These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g, labeled sheep anti-mouse antibodies) that specifically bind to the anti-TIR antibodies.

Other, assay formats include liposome immunoassays (LIA), which use 10 liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see Monroe et al., Amer. Clin. Prod. Rev., 5:34-41 (1986)). E. Reduction of non-specific binding 15 One of skill in the art will appreciate that it is often desirable to minimize non specific binding in immunoassays. Particularly, where the assay involves an antigen or antibody immobilized on a solid substrate it is desirable to minimize the amount of non-specific binding to the substrate. Means of reducing such non-specific binding are well known to those of skill in the art. Typically, this technique involves coating the 20 substrate with a proteinaceous composition. In particular, protein compositions such as bovine serum albumin (BSA), nonfat powdered milk, and gelatin are widely used with powdered milk being most preferred. 25 F. Labels

The particular label or detectable group used in the assay is not a critical aspect of the invention, as long as it does not significantly interfere with the specific binding of the antibody used in the assay. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well 30 developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical, or chemical means. Useful labels in the present invention include magnetic 110 beads (e.g., DYNABEADSTM), fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g, 3H, 125I, 14C, 35S), enzymes (e.g., horseradish peroxidase, alkaline phosphates and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic beads (e.g, polystyrene, polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instrumentation, and disposal provisions.

Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g, biotin) is covalently bound to the molecule. The ligand then binds to another molecules (e.g, streptavidin) molecule, which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. The ligands and their targets can be used in any suitable combination with antibodies that recognize a T1R polypeptide, or secondary antibodies that recognize anti-TIR.

The molecules can also be conjugated directly to signal generating compounds, e.g, by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidotases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.

Chemiluminescent compounds include luciferin, and 2,3-dihydrophthalazinediones, e.g, luminol. For a review of various labeling or signal producing systems that may be used, see U.S. Patent No. 4,391,904.

Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge-coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the 111

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enzyme and detecting the resulting reaction product. Finally simple colorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead. 5 Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection. 10

Detection of Modulators

Compositions and methods for determining whether a test compound specifically binds to a T1R receptor of the invention, both in vitro and in vivo, are described below. Many aspects of cell physiology can be monitored to assess the effect 15 of ligand binding to a T1R polypeptide of the invention. These assays may be performed on intact cells expressing a chemosensory receptor, on permeabilized cells, or on membrane fractions produced by standard methods or in vitro de novo synthesized proteins.

In vivo, taste receptors bind tastants and initiate the transduction of chemical 20 stimuli into electrical signals. An activated or inhibited G protein will in turn alter the properties of target enzymes, channels, and other effector proteins. Some examples are the activation of cGMP phosphodiesterase by transducin in the visual system, adenylate cyclase by the stimulatory G protein, phospholipase C by Gq and other cognate G proteins, and modulation of diverse channels by Gi and other G proteins. Downstream 25 consequences can also be examined such as generation of diacyl glycerol and IP3 by phospholipase C, and in turn, for calcium mobilization by IP3.

The T1R proteins or polypeptides of the assay will preferably be selected from a polypeptide having the T1R polypeptide sequence selected from those disclosed in Example 1, or fragments or conservatively modified variants thereof. Optionally, the 30 fragments and variants can be antigenic fragments and variants which bind to an anti-T1R antibody. Optionally, the fragments and variants can bind to or are activated by sweeteners or umami tastants. 112

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Alternatively, the T1R proteins or polypeptides of the assay can be derived from a eukaryotic host cell and can include an amino acid subsequence having amino acid sequence identity to the T1R polypeptides disclosed in Example 1, or fragments or conservatively modified variants thereof Generally, the amino acid sequence identity 5 will be at least 35 to 50%, or optionally 75%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%. Optionally, the T1R proteins or polypeptides of the assays can comprise a domain of a T1R protein, such as an extracellular domain, transmembrane region, transmembrane domain, cytoplasmic domain, ligand-binding domain, and the like. Further, as described above, the T1R protein or a domain thereof can be covalently 10 linked to a heterologous protein to create a chimeric protein used in the assays described herein.

Modulators of T1R receptor activity are tested using T1R proteins or polypeptides as described above, either recombinant or naturally occurring. The T1R proteins or polypeptides can be isolated, co-expressed in a cell, co-expressed in a 15 membrane derived from a cell, co-expressed in tissue or in an animal, either recombinant or naturally occurring. For example, tongue slices, dissociated cells from a tongue, transformed cells, or membranes can be used. Modulation can be tested using one of the in vitro or in vivo assays described herein.

For example, as disclosed in the experiment examples infra, it has been 20 discovered that certain 5 nucleotides, e.g., 5 IMP or 5 GMP, enhance the activity of L-glutamate to activate the umami taste receptor, or block the activation of the umami taste receptor by umami taste stimuli such as L-glutamate and L-aspartate. 25 1. In vitro binding assays

Taste transduction can also be examined in vitro with soluble or solid state reactions, using the T1R polypeptides of the invention. In a particular embodiment, T1R ligand-binding domains can be used in vitro in soluble or solid state reactions to 30 assay for ligand binding.

For instance, the T1R N-terminal domain is predicted to be involved in ligand binding. More particularly, the TIRs belong to a GPCR sub-family that is characterized by large, approximately 600 amino acid, extracellular N-terminal 113

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segments. These N-terminal segments are thought to form the ligand-binding domains, and are therefore useful in biochemical assays to identify T1R agonists and antagonists. It is possible that the ligand-binding domain may be formed by additional portions of the extracellular domain, such as the extracellular loops of the transmembrane domain. 5 In vitro binding assays have been used with other GPCRs that are related to the TIRs, such as the metabotropic glutamate receptors (see, e.g., Han and Hampson, J. Bioi Chem. 274:10008-10013 (1999)). These assays might involve displacing a radioactively or fluorescently labeled ligand, measuring changes in intrinsic fluorescence or changes in proteolytic susceptibility, etc. 10 Ligand binding to a hetero-multimeric complex of T1R polypeptides of the invention can be tested in solution, in a biiayer membrane, optionally attached to a solid phase, in a lipid monolayer, or in vesicles. Binding of a modulator can be tested using, e.g, changes in spectroscopic characteristics (e.g, fluorescence, absorbence, refractive index) hydrodynamic (e.g, shape), chromatographic, or solubility properties. 15 [0207] In another embodiment of the invention, a GTPy35S assay may be used. As described above, upon activation of a GPCR, the Ga subunit of the G protein complex is stimulated to exchange bound GDP for GTP. Ligand-mediated stimulation of G protein exchange activity can be measured in a biochemical assay measuring the binding of added radioactively labeled GTPy35S to the G protein in the presence of a 20 putative ligand. Typically, membranes containing the chemosensory receptor of interest are mixed with a complex of G proteins. Potential inhibitors and/or activators and GTPy35S are added to the assay, and binding of GTPy35S to the G protein is measured. Binding can be measured by liquid scintillation counting or by any other means known in the art, including scintillation proximity assays (SPA). In other assays 25 formats, fluorescently labeled GTPyS can be utilized. 2. Fluorescence Polarization Assays

In another embodiment, Fluorescence Polarization (“FP”) based assays may be used to detect and monitor ligand binding. Fluorescence polarization is a versatile 30 laboratory technique for measuring equilibrium binding, nucleic acid hybridization, and enzymatic activity. Fluorescence polarization assays are homogeneous in that they do not require a separation step such as centrifugation, filtration, chromatography, precipitation, or electrophoresis. These assays are done in real time, directly in solution 114 and do not require an immobilized phase. Polarization values can be measured repeatedly and after the addition of reagents since measuring the polarization is rapid and does not destroy the sample. Generally, this technique can be used to measure polarization values of fluorophores from low pi comolar to micro mo lar levels. This section describes how fluorescence polarization can be used in a simple and quantitative way to measure the binding of ligands to the T1R polypeptides of the invention.

When a fluorescently labeled molecule is excited with plane-polarized light, it emits light that has a degree of polarization that is inversely proportional to its molecular rotation. Large fluorescently labeled molecules remain relatively stationary during the excited state (4 nanoseconds in the case of fluorescein) and the polarization of the light remains relatively constant between excitation and emission. Small fluorescently labeled molecules rotate rapidly during the excited state and the polarization changes significantly between excitation and emission. Therefore, small molecules have low polarization values and large molecules have high polarization values. For example, a single-stranded fluorescein-labeled oligonucleotide has a relatively low polarization value but when it is hybridized to a complementary strand, it has a higher polarization value. When using FP to detect and monitor tastant-binding which may activate or inhibit the chemosensory receptors of the invention, fluorescence-labeled tastants or auto-fluorescent tastants may be used.

Fluorescence polarization (P) is defined as:

pJntn-IntL lntu + IntL

Where Π is the intensity of the emission light parallel to the excitation light plane and Int 1 is the intensity of the emission light perpendicular to the excitation light plane. P, being a ratio of light intensities, is a dimensionless number. For example, the Beacon ® and Beacon 2000 ™ System may be used in connection with these assays. Such systems typically express polarization in millipolarization units (1 Polarization Unit =1000 mP Units).

The relationship between molecular rotation and size is described by the Perrin equation and the reader is referred to Jolley, Μ. E. (1991) in Journal of Analytical Toxicology, pp. 236-240, which gives a thorough explanation of this equation. Summarily, the Perrin equation states that polarization is directly proportional to the 115

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10 15 rotational relaxation time, the time that it takes a molecule to rotate through an angle of approximately 68.5° Rotational relaxation time is related to viscosity (η), absolute temperature (T), molecular volume (V), and the gas constant (R) by the following equation:

Rotational Re taxation Time =

RT

The rotational relaxation time is small (- 1 nanosecond) for small molecules (e.g. fluorescein) and large (« 100 nanoseconds) for large molecules (e.g. immunoglobulins). If viscosity and temperature are held constant, rotational relaxation time, and therefore polarization, is directly related to the molecular volume. Changes in molecular volume may be due to interactions with other molecules, dissociation, polymerization, degradation, hybridization, or conformational changes of the fluorescently labeled molecule. For example, fluorescence polarization has been used to measure enzymatic cleavage of large fluorescein labeled polymers by proteases, DNases, and RNases. It also has been used to measure equilibrium binding for protein/protein interactions, antibody/antigen binding, and protein/DNA binding. A. Solid state and soluble high throughput assays In yet another embodiment, the invention provides soluble assays using a

hetero-oligomeric T1R polypeptide complex; or a cell or tissue co-expressing T1R 20 polypeptides. Preferably, the cell will comprise a cell line that stably co-expresses a functional T1R1/T1R3 (umami) taste receptor or T1R2/T1R3 (sweet) taste receptor. In another embodiment, the invention provides solid phase based in vitro assays in a high throughput format, where the T1R polypeptides, or cell or tissue expressing the T IR polypeptides is attached to a solid phase substrate or a taste stimulating compound and 25 contacted with a T1R receptor, and binding detected using an appropriate tag or antibody raised against the T1R receptor.

In the high throughput assays of the invention, it is possible to screen up to several thousand different modulators or ligands in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential 30 modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (e.g, 96) modulators. If 1536 well plates are used, then a single plate can 116

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easily assay from about 1000 to about 1500 different compounds. It is also possible to assay multiple compounds in each plate well. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 different compounds is possible using the integrated systems of the invention. More recently, microfluidic 5 approaches to reagent manipulation have been developed.

The molecule of interest can be bound to the solid state component, directly or indirectly, via covalent or non-covalent linkage, e.g, via a tag. The tag can be any of a variety of components. In general, a molecule which binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest (e.g, the taste transduction 10 molecule of interest) is attached to the solid support by interaction of the tag and the tag binder. A number of tags and tag binders can be used, based upon known molecular interactions well described in the literature. For example, where a tag has a natural binder, for example, biotin, protein A, or protein G, it can be used in conjunction with 15 appropriate tag binders (avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.). Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis MO).

Similarly, any haptenic or antigenic compound can be used in combination with 20 an appropriate antibody to form a tag/tag binder pair. Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature. For example, in one common configuration, the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody. In addition to antibody-antigen interactions, receptor-ligand interactions are also appropriate as tag 25 and tag-binder pairs. For example, agonists and antagonists of cell membrane receptors (e.g, cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherein family, the integrin family, the selectin family, and the like; see, e.g, Pigott &amp;amp; Power, The Adhesion Molecule Facts Book I (1993)). 30 Similarly, toxins and venoms, viral epitopes, hormones (e.g, opiates, steroids, etc.), intracellular receptors (e.g, which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides), drugs, lectins, 117

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sugars, nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.

Synthetic polymers, such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, 5 polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.

Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly gly sequences of between about 5 10 and 200 amino acids. Such flexible linkers are known to persons of skill in the art. For example, poly(ethelyne glycol) linkers are available from Shearwater Polymers, Inc. Huntsville, Alabama. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.

Tag binders are fixed to solid substrates using any of a variety of methods 15 currently available. Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder. For example, groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes and 20 hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The constitutive of such solid phase biopolymer arrays is well described in the literature. See, e.g., Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963) (describing solid phase synthesis of, e.g, peptides); Geysen et ai, J. Immun. Meth., 102:259-274 (1987) (describing synthesis of solid phase components on pins); Frank &amp;amp; 25 Doring, Tetrahedron, 44:60316040 (1988) (describing synthesis of various peptide sequences on cellulose disks); Fodor etal., Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry, 39(4):718-719 (1993); and Kozal et al., Nature Medicine, 2(7):753759 (1996) (all describing arrays of biopolymers fixed to solid substrates). Non-chemical approaches for fixing tag binders to substrates include other common 30 methods, such as heat, cross-linking by UV radiation, and the like. 118

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3. Cell-based assays

In a preferred embodiment of treatment, a combination of T1R proteins or polypeptides are transiently or stably co-expressed in a eukaryotic cell either in unmodified forms or as chimeric, variant or truncated receptors with or preferably 5 without a heterologous, chaperone sequence that facilitates its maturation and targeting through the secretory pathway. Such T1R polypeptides can be expressed in any eukaryotic cell, such as HEK-293 cells. Preferably, the cells comprise a functional G protein, e.g., Gal5 or the chimeric G protein previously identified, or another G protein that is capable of coupling the chimeric receptor to an intracellular signaling pathway 10 or to a signaling protein such as phospholipase C. Also, preferably a cell will be produced that stably co-expresses T1R1/T1R3 or T1R2/T1R3 as such cells have been found (as shown in the experimental examples) to exhibit enhanced responses to taste stimuli (relation to cells that transiently express the same T1R combination).

Activation of T1R receptors in such cells can be detected using any standard method, 15 such as by detecting changes in intracellular calcium by detecting Fluo-4 dependent fluorescence in the cell. Such an assay is the basis of the experimental findings presented in this application.

Activated GPCR receptors often are substrates for kinases that phosphorylate the C-terminal tail of the receptor (and possibly other sites as well). Thus, activators 20 will promote the transfer of 32P from radiolabeled ATP to the receptor, which can be assayed with a scintillation counter. The phosphorylation of the C-terminal tail will promote the binding of arrestin-like proteins and will interfere with the binding of G proteins. For a general review of GPCR signal transduction and methods of assaying signal transduction, see, e.g, Methods in Enzymology, vols. 237 and 238 (1994) and 25 volume 96 (1983); Bourne et al., Nature, 10:349:117-27 (1991); Bourne et al., Nature, 348:125-32 (1990); Pitcher et ai, Annu. Rev. Biochem., 61.653-92 (1998).

T1R modulation may be assayed by comparing the response of T1R polypeptides treated with a putative T1R modulator to the response of an untreated control sample or a sample containing a known “positive” control. Such putative T1R 30 modulators can include molecules that either inhibit or activate T1R polypeptide activity. In one embodiment, control samples (untreated with activators or inhibitors) are assigned a relative T1R activity value of 100. Inhibition of a T1R polypeptide is achieved when the T1R activity value relative to the control is about 90%, optionally 119

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50%, optionally 25-0%. Activation of a TI R polypeptide is achieved when the T1R activity value relative to the control is 110%, optionally 150%, 200-500%, or 1000-2000%.

Changes in ion flux may be assessed by determining changes in ionic

5 polarization (i.e., electrical potential) of the cell or membrane expressing a T1R polypeptide. One means to determine changes in cellular polarization is by measuring changes in current (thereby measuring changes in polarization) with voltage-clamp and patch-clamp techniques (see, e.g., the “cell-attached” mode, the “inside-out” mode, and the “whole cell” mode, e.g., Ackerman et al, New Engl J Med., 336:1575-1595 10 (1997)). Whole cell currents are conveniently determined using the standard. Other known assays include: radiolabeled ion flux assays and fluorescence assays using voltage-sensitive dyes (see, e.g., Vestergarrd-Bogind et al, J. Membrane Biol, 88:67-75 (1988); Gonzales &amp;amp; Tsien, Chem. Biol, 4:269277 (1997); Daniel et al, J. Pharmacol. Meth., 25:185-193 (1991); Holevinsky etal, J. Membrane Biology, 15 137:59-70 (1994)).

The effects of the test compounds upon the function of the polypeptides can be measured by examining any of the parameters described above. Any suitable physiological change that affects GPCR activity can be used to assess the influence of a test compound on the polypeptides of this invention. When the functional 20 consequences are determined using intact cells or animals, one can also measure a variety of effects such as transmitter release, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as Ca2+, IP3, cGMP, or cAMP. 25 Preferred assays for GPCRs include cells that are loaded with ion or voltage sensitive dyes to report receptor activity. Assays for determining activity of such receptors can also use known agonists and antagonists for other G protein-coupled receptors as controls to assess activity of tested compounds. In assays for identifying modulatory compounds (e.g., agonists, antagonists), changes in the level of ions in the 30 cytoplasm or membrane voltage will be monitored using an ion sensitive or membrane voltage fluorescent indicator, respectively. Among the ion-sensitive indicators and voltage probes that may be employed are those disclosed in the Molecular Probes 1997 Catalog. For G protein-coupled receptors, promiscuous G proteins such as Gal5 and 120

Gal 6 can be used in the assay of choice (Wilkie et al., Proc. Nat 7 Acad Sci., 88:10049-10053 (1991)).

Receptor activation initiates subsequent intracellular events, e.g., increases in second messengers. Activation of some G protein-coupled receptors stimulates the formation of inositol triphosphate (1P3) through phospholipase C-mediated hydrolysis of phosphatidylinositol (Berridge &amp;amp; Irvine, Nature, 312:315-21 (1984)). IP3 in turn stimulates the release of intracellular calcium ion stores. Thus, a change in cytoplasmic calcium ion levels, or a change in second messenger levels such as IP3 can be used to assess G protein-coupled receptor function. Cells expressing such G protein-coupled receptors may exhibit increased cytoplasmic calcium levels as a result of contribution from both calcium release from intracellular stores and extracellular calcium entry via plasma membrane ion channels.

In a preferred embodiment, T1R polypeptide activity is measured by stably or transiently co-expressing T1R genes, preferably stably, in a heterologous cell with a promiscuous G protein that links the receptor to a phospholipase C signal transduction pathway (see Offermanns &amp;amp; Simon, J. Biol. Chem., 270:15175-15180 (1995)). In a preferred embodiment, the cell line is HEK-293 (which does not normally express T1R genes) and the promiscuous G protein is Gal5 (Offermanns &amp;amp; Simon, supra).

I

Modulation of taste transduction is assayed by measuring changes in intracellular Ca levels, which change in response to modulation of the T1R signal transduction pathway via administration of a molecule that associates with T1R polypeptides. Changes in Ca2+ levels are optionally measured using fluorescent Ca2+ indicator dyes and fluorometric imaging.

In another embodiment, phosphatidyl inositol (PI) hydrolysis can be analyzed according to U.S. Patent 5,436,128, herein incorporated by reference. Briefly, the assay involves labeling of cells with 3H-myoinositol for 48 or more hrs. The labeled cells are treated with a test compound for one hour. The treated cells are lysed and extracted in chloroform-methanol-water after which the inositol phosphates were separated by ion exchange chromatography and quantified by scintillation counting. Fold stimulation is determined by calculating the ratio of cpm in the presence of agonist, to cpm in the presence of buffer control. Likewise, fold inhibition is determined by calculating the ratio of cpm in the presence of antagonist, to cpm in the presence of buffer control (which may or may not contain an agonist). .121

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Other receptor assays can involve determining the level of intracellular cyclic nucleotides, e.g., cAMP or cGMP. In cases where activation of the receptor results in a decrease in cyclic nucleotide levels, it may be preferable to expose the cells to agents that increase intracellular cyclic nucleotide levels, e.g, forskolin, prior to adding a 5 receptor-activating compound to the cells in the assay. In one embodiment, the changes in intracellular cAMP or cGMP can be measured using immunoassays. The method described in Offermanns &amp;amp; Simon, J. Bio. Chem., 270:15175-15180 (1995), may be used to determine the level of cAMP. Also, the method described in Felley-Bosco et al.,Am. J. Resp. Cel! and Mol. Biol., 11:159-164 (1994), may be used to determine the 10 level of cGMP. Further, an assay kit for measuring cAMP and/or cGMP is described in U.S. Patent 4,115,538, herein incorporated by reference.

In another embodiment, transcription levels can be measured to assess the effects of a test compound on signal transduction. A host cell containing T1R polypeptides of interest is contacted with a test compound for a sufficient time to effect 15 any interactions, and then the level of gene expression is measured. The amount of time to effect such interactions may be empirically determined, such as by running a time course and measuring the level of transcription as a function of time. The amount of transcription may be measured by using any method known to those of skill in the art to be suitable. For example, mRNA expression of the protein of interest may be 20 detected using northern blots or their polypeptide products may be identified using immunoassays. Alternatively, transcription based assays using reporter gene may be used as described in U.S. Patent 5,436,128, herein incorporated by reference. The reporter genes can be, e.g, chloramphenicol acetyltransferase, luciferase, beta-galactosidase beta-lactamase and alkaline phosphatase. Furthermore, the protein 25 of interest can be used as an indirect reporter via attachment to a second reporter such as green fluorescent protein (see, e.g., Mistili &amp;amp; Spector, Nature Biotechnology, 15:961-964 (1997)).

The amount of transcription is then compared to the amount of transcription in either the same cell in the absence of the test compound, or it may be compared with

30 the amount of transcription in a substantially identical cell that lacks the T1R polypeptide(s) of interest. A substantially identical cell may be derived from the same cells from which the recombinant cell was prepared but which had not been modified by introduction of heterologous DNA. Any difference in the amount of transcription 122 indicates that the test compound has in some manner altered the activity of the T1R polypeptides of interest. 4. Transgenic non-human animals expressing chemosensory receptors

Non-human animals expressing a combination of T1R taste receptor sequences of the invention can also be used for receptor assays. Such expression can be used to determine whether a test compound specifically binds to a mammalian taste transmembrane receptor complex in vivo by contacting a non-human animal stably or transiently transfected with nucleic acids encoding chemosensory receptors or ligandbinding regions thereof with a test compound and determining whether the animal reacts to the test compound by specifically binding to the receptor polypeptide complex.

Animals transfected or infected with the vectors ofthe invention are particularly useful for assays to identify and characterize taste stimuli that can bind to a specific or sets of receptors. Such vector-infected animals expressing human taste receptor sequences can be used for in vivo screening of taste stimuli and their effect on, e.g., cell physiology (e.g., on taste neurons), on the CNS, or behavior. Alternatively, stable cell lines that express a T1R or combination thereof, can be used as nucleic transfer donors to produced cloned transgenic animals that stably express a particular T1R or combination. Methods of using nucleic transfer to produce cloned animals that express a desired heterologous DNA are the subject of several issued U.S. patents granted to the University of Massachusetts (licensed to Advanced Cell Technology, Inc.) and Roslin Institute (licensed to Geron Corp.).

Means to infect/express the nucleic acids and vectors, either individually or as libraries, are well known in the art. A variety of individual cell, organ, or whole animal parameters can be measured by a variety of means. The T1R sequences of the invention can be for example co-expressed in animal taste tissues by delivery with an infecting agent, e.g., adenovirus expression vector.

The endogenous taste receptor genes can remain functional and wild-type (native) activity can still be present. In other situations, where it is desirable that all taste receptor activity is by the introduced exogenous hybrid receptor, use of a knockout line is preferred. Methods for the constitutive of non-human transgenic 123 animals, particularly transgenic mice, and the selection and preparation of recombinant constructs for generating transformed cells are well known in the art.

Constitutive of a “knockout” cell and animal is based on the premise that the level of expression of a particular gene in a mammalian cell can be decreased or completely abrogated by introducing into the genome a new DNA sequence that serves to interrupt some portion of the DNA sequence of the gene to be suppressed. Also, “gene trap insertion” can be used to disrupt a host gene, and mouse embryonic stem (ES) cells can be used to produce knockout transgenic animals (see, e.g., Holzschu, Transgenic Res 6:97-106 (1997)). The insertion of the exogenous is typically by homologous recombination between complementary nucleic acid sequences. The exogenous sequence is some portion of the target gene to be modified, such as exonic, intronic or transcriptional regulatory sequences, or any genomic sequence which is able to affect the level of the target gene’s expression; or a combination thereof. Gene targeting via homologous recombination in pluripotential embryonic stem cells allows one to modify precisely the genomic sequence of interest. Any technique can be used to create, screen for, propagate, a knockout animal, e.g, see Bijvoet, Hum. Mol. Genet. 7:53-62 (1998); Moreadith, J. Mol. Med. 75:208-216 (1997); Tojo, Cytotechnology 19:161-165 (1995); Methods Mol. Biol. 48:167-184 (1995); Longo,

Transgenic Res. 6:321-328 (1997); U.S. Patents Nos. 5,616,491; 5,464,764; 5,631,153; 5,487,992; 5,627,059; 5,272,071; WO 91/09955; WO93/09222; WO 96/29411; WO 95/31560; WO 91/12650.

The nucleic acids of the invention can also be used as reagents to produce “knockout” human cells and their progeny. Likewise, the nucleic acids of the invention can also be used as reagents to produce “knock-ins” in mice. The human or rat T1R gene sequences can replace the orthologous T1R in the mouse genome. In this way, a mouse expressing a human or rat T1R is produced. This mouse can then be used to analyze the function of human or rat TIRs, and to identify ligands for such TIRs. a. Modulators

The compounds tested as modulators of a T1R family member can be any small chemical compound, or a biological entity, such as a protein, nucleic acid or lipid. Examples thereof include 51 IMP and 51 GMP. Essentially any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds that are soluble in aqueous solutions are tested. Assays can be 124 designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source; these assays are typically run in parallel (e.g, in microtiter formats on microtiter plates in robotic assays). It will be appreciated that chemical libraries can be synthesized by one of many chemical reactions (e.g. Senomyx proprietary chemistries). Additionally, there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma-Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and the like.

In one preferred embodiment, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential taste affecting compounds (potential modulator or ligand compounds). Such “combinatorial chemical libraries” or “ligand libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional “lead compounds” or can themselves be used as potential or actual taste modulators.

Preferably, such libraries will be screened against cells or cell lines that stably express a T1R or combination of TIRs, i.e. T1R1/T1R3 or T1R2/T1R3 and preferably a suitable G protein, e.g. Gai5· As shown in the examples infra, such stable cell lines exhibit very pronounced responses to taste stimuli, e.g. umami or sweet taste stimuli. However, cells and cell lines that transiently express one or more TIRs may also be used in such assays. A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical “building blocks” such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Thousands to millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.

Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g, U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. 125

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Res., 37:487-493 (1991) and Houghton et al., Nature, 354:84-88 (1991)). Other chemistries for generating chemically diverse libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g, PCT Publication No. WO 91/19735), encoded peptides (e.g, PCT Publication WO 93/20242), random 5 bio-oligomers (e.g, PCT Publication No. WO 92/00091), benzodiazepines (e.g, U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs etal., Proc. Nat. Acad Sci., 90:6909-6913 (1993)), vinylogous polypeptides (Hagihara etal., J. Amer. Chem. Soc., 114:6568 (1992)), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al.,J. Amer. Chem. Soc., 114:9217-9218 10 (1992)), analogous organic syntheses of small compound libraries (Chen et al.,J. Amer.

Chem. Soc., 116:2661 (1994)), oligocarbamates (Cho etal., Science, 261:1303 (1993)), peptidyl phosphonates (Campbell et al., J. Org. Chem., 59:658 (1994)), nucleic acid libraries (Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (U.S. Patent 5,539,083), antibody libraries (Vaughn et al., Nature Biotechnology, 15 14(3):309-314 (1996) and PCT/US96/10287), carbohydrate libraries (Liang etal.,

Science, 274:1520-1522 (1996) and U.S. Patent 5,593,853), small organic molecule libraries (benzodiazepines, Baum, C&amp;amp;EN, Jan 18, page 33 (1993); thiazolidinones and metathiazanones, U.S. Patent 5,549,974; pynrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent 5,506,337; benzodiazepines, 5,288,514, 20 and the like).

Devices for the preparation of combinatorial libraries are commercially available (see, e.g, 357 MPS, 390 MPS (Advanced Chem Tech, Louisville KY), Symphony (Rainin, Woburn, MA), 433A (Applied Biosystems, Foster City, CA), 9050 Plus (Millipore, Bedford, MA)). In addition, numerous combinatorial libraries are 25 themselves commercially available (see, e.g, ComGenex, Princeton, NJ; Tripos, Inc.,

St. Louis, MO; 3D Pharmaceuticals, Exton, PA; Martek Biosciences; Columbia, MD; etc.).

In one aspect of the invention, the T1R modulators can be used in any food product, confectionery, pharmaceutical composition, or ingredient thereof to thereby 30 modulate the taste of the product, composition, or ingredient in a desired manner. For instance, T1R modulators that enhance sweet taste sensation can be added to sweeten a product or composition; T1R modulators that enhance umami taste sensation can be 126 added to foods to increase savory tastes. Alternatively, T1R antagonists can be used to block sweet and/or umami taste. b. Kits T1R genes and their homologs are useful tools for identifying chemosensory receptor cells, for forensics and paternity determinations, and for examining taste transduction. T1R family member-specific reagents that specifically hybridize to TIR nucleic acids, such as TIR probes and primers, and TIR specific reagents that specifically bind to a TIR polypeptide, e.g., TIR antibodies are used to examine taste cell expression and taste transduction regulation.

Nucleic acid assays for the presence of DNA and RNA for a TIR family member in a sample include numerous techniques are known to those skilled in the art, such as southern analysis, northern analysis, dot blots, RNase protection, SI analysis, amplification techniques such as PCR, and in situ hybridization. In in situ hybridization, for example, the target nucleic acid is liberated from its cellular surroundings in such as to be available for hybridization within the cell while preserving the cellular morphology for subsequent interpretation and analysis. The following articles provide an overview of the art of in situ hybridization: Singer et al., Biotechniques, 4:230250 (1986); Haase et al, Methods in Virology, vol. VII, pp. 189-226 (1984); and Nucleic Acid Hybridization: A Practical Approach (Names et al., eds. 1987). In addition, a TIR polypeptide can be detected with the various immunoassay techniques described above. The test sample is typically compared to both a positive control (e.g, a sample expressing a recombinant TIR polypeptide) and a negative control.

The present invention also provides for kits for screening for modulators of TIR family members. Such kits can be prepared from readily available materials and reagents. For example, such kits can comprise any one or more of the following materials: TIR nucleic acids or proteins, reaction tubes, and instructions for testing TIR activity. Optionally, the kit contains a biologically active TIR receptor or cell line that stably or transiently expresses a biologically active TIR containing taste receptor. A wide variety of kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user. 127

EXAMPLES

While the invention has been described in detail supra, the following examples are provided to illustrate preferred embodiments. These examples are intended to be illustrative and not limitative of the scope of the invention.

In the protein sequences presented herein, the one-letter code X or Xaa refers to any of the twenty common amino acid residues. In the DNA sequences presented herein, the one letter codes N or n refers to any of the of the four common nucleotide bases, A, T, C, or G.

Example 1

Production of Intronless hTIR Expression Constructs

Intronless hTIR expression constructs were cloned by a combination of cDNA-based and genomic DNA-based methods. To generate the full-length hTIRl expression construct, two 5’ coding exons identified in a cloned hTIRl interval (accession # ALI 59177) were combined by PCR-overlap, and then joined to a 5’-truncated testis cDNA clone. The hTlR2 expression construct was generated from a partially sequenced hTlR2 genomic interval. Two missing hTlR2 5’ exons were identified by screening shotgun libraries of the cloned genomic interval using probes derived from the corresponding rat coding sequence. Coding exons were then combined by PCR-overlap to produce the full-length expression construct. The hT lR3 expression construct was generated by PCR-overlap from a sequenced hTlR3 genomic interval (accession # AL139287). Rat T1R3 was isolated from a rat taste tissue-derived cDNA library using an rTlR3 exon fragment generated by hTlR3-based degenerate PCR. The partial hTIRl cDNA, rTlR2 cDNA, and partial hT!R2 genomic sequences were obtained from Dr. Charles Zuker (University of California, San Diego).

The nucleic acid and amino acid sequences for the above-identified T1R cloned sequences as well as other full-length and partial T1R sequences are set forth in the sequence listing.

Also, the following conceptual translations, which correspond to the C-termini of two orthologous pairs of fish TIRs, are derived from unpublished genomic sequence fragments and provided. Fugu TIRA was derived from accession 'scaffold 164'; Fugu T1RB was derived from accession LPC61711; Tetradon TIRA was derived from 128 accession AL226735; Tetradon T1RB was derived from accession AL222381. Ambiguities in the conceptual translations ('X') result from ambiguities in database sequences. These sequences can be found in the sequence listing.

Additionally, the accession number and reference citations relating to mouse and rat TIRs and allelic variants thereof in the public domain are is set forth below: rTIRl (Accession # AAD18069) (Hoon et at, Cell 96 (4): 541-51 (1999)); rTlR2 (Accession # AAD18070) (Hoon et at, Cell 96(4): 541-59 (1999)); mTIRl (Accession # AAK39437); mTlR2 (Accession #AAK 39438); mTlR3 (Accession AAK 55537) (Max et al., Nat. Genet. 28(1): 58-63 (2001)); rTIRl (Accession # AAK7092) (Li et at, Mamm. Genome (12(1): 13-16 (2001)); mTIRl (Accession # NP 114073); mTIRl (Accession # AAK07091) (Li et at, Mamm. Genome (121):13-16 (2001)); rTlR2 (Accession # AAD18070) (Hoon et at, Cell 9664): 541-551 (1999)); mTlR2 (Accession # NPI 14079); mTlR3 (Accession# AAK39436); mTlR3 (Accession # BAB47181); (Kitagawa et at, Biochem. Biophys. Res. Comm. 283(1):236-42 (2001)); mTlR3 (Accession #NP114078); mTlR3 (Accession # AAK55536) (Max et at, Nat Genet. 28(1):58-63 (2001)); and mTlR3 (Accession No. AAK01937).

Example 2

Sequence Alignment of Human and Rat TIRs

Cloned T1R sequences selected from those identified above were aligned against the corresponding rat TIRs. As shown in Figure 1, human T1R1, human T1R2 and human T1R3 and rat T1R3 were aligned with previously described TIRs (rTIRl having Accession # AAD18069 and rTlR2 having Accession # AAD18070), the rat mGluRl metabotropic, glutamate receptor (Accession # P23385); and the human calcium-sensing receptor (Accession #P41180). For clarity of the comparison, the mGluRl and calcium-sensing receptor C-termini are truncated. The seven potential transmembrane segments are boxed in blue. Residues that contact the glutamate side-chain carbutylate in the mGluRl crystal structure are boxed in red, and residues that contact the glutamate α-amino acid moiety are boxed in green. The mGluRl and calcium-sensing receptor cysteine residues implicated in intersubunit disulfide-based formation are circled in purple. These cysteines are not conserved in T1R1 and T1R2, 129 but are located in a degraded region of the alignment that contains a potentially analogous T1R3 cysteine residue, also circled.

Example 3

Demonstration by RT-PCR that hT!R2 and hTlR3 are expressed in taste tissue

As shown in Figure 2, hTlR2 and hTlR3 are expressed in taste tissue: expression of both genes can be detected by RT-PCR from resected human circumvallate papillae.

Example 4

Methods for Heterologous Expression of TIRs in Heterologous Cells

An HEK-293 derivative (Chandrashekar et al., Cell 100(6): 703-11 (2000)), which stably expresses Gal5, was grown and maintained at 37°C in Dulbecco’s Modified Eagle Medium (DMEM, Gibco BRL) supplemented with 10% FBS, MEM non-essential amino acids (Gibco BRL), and 3 rtg/ml blasticidin. For calcium-imaging experiments, cells were first seeded onto 24-well tissue-culture plates (approximately 0.1 million cells per well), and transfected by lipofection with Mirus Transit-293 (PanVera). To minimize glutamate-induced and glucose-induced desensitization, supplemented DMEM was replaced with low-glucose DMEM/GlutaMAX (Gibco BRL) approximately 24 hours after transfection. 24 hours later, cells were loaded with the calcium dye Fluo-4 (Molecular Probes), 3μΜ in Dulbecco’s PBS buffer (DPBS, GibcoBRL), for 1.5 hours at room temperature. After replacement with 250μ1 DPBS, stimulation was performed at room temperature by addition of 200μ1 DPBS supplemented with taste stimuli. Calcium mobilization was monitored on a Axiovert SI00 TV microscope (Zeiss) using Imaging Workbench 4.0 software (Axon). T1R1/T1R3 and T1R2/T1R3 responses were strikingly transient - calcium increases rarely persisted longer than 15 seconds - and asynchronous. The number of responding cells was thus relatively constant over time; therefore, cell responses were quantitated by manually counting the number of responding cells at a fixed time point, typically 30 seconds after stimulus addition. 130

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Example 5

Human T1R2/T1R3 functions as a sweet taste receptor HEK cells stably expressing Gal 5 were transiently transfected with human T1R2, T1R3 and T1R2/T1R3, and assayed for increases in intracellular calcium in 5 response to increasing concentrations of sucrose (Figure 3(a)). Also, T1R2/T1R3 dose responses were determined for several sweet taste stimuli (Figure 3(b)). The maximal percentage of responding cells was different for different sweeteners, ranging from 10-30%. For clarity, dose responses were normalized to the maximal percentage of responding cells. The values in Figure 3 represent the mean + s.e. of four independent 10 responses. X-axis circles mark psychophysical detection thresholds determined by taste testing. Gurmarin (50-fold dilution of a filtered 10g/l Gyrrtnema sylvestre aqueous extract) inhibited the response of T1R2/T1R3 to 250 mM sucrose, but not the response of endogenous P2-adrenergic receptor to 20 μΜ isoproterenol (Figure 3(b)). Figure 3(c) contains the normalized response of T1R2/T1R3 co-expressing cell lines to 15 different sweeteners(sucrose, aspartame, D-tryptophan and saccharin). 131

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Example 6

Rat T1R2/T1R3 also functions as a sweet taste receptor HEK cells stably expressing Gal5 were transiently transfected with 5 hTlR2/hTlR3, rTlR2/rTlR3, hTlR2/rTlR3, and rTlR2/hTlR3. These transfected cells were then assayed for increased intracellular calcium in response to 350 mM sucrose, 25 mM tryptophan, 15 mM aspartame, and 0,05% of monellin. The results with sucrose and aspartame are contained in Figure 4 and indicate that rTlR2/rTlR3 also functions as a sweet taste receptor. Also, these results suggest that T1R2 may 10 control T1R2/T1R3 ligand specificity.

Example 7 T1R2/T1R3 responses using an automated fluorescence based assay HEK cells stably expressing Gal5 were transiently transfected with hTlR2 and 15 hTlR3. These cells were loaded with the calcium dye Fluo-4, and their responses to a sweetener measured using a fluorescence plate reader. Figure 5 contains cyclamate (12.5 mM) responses for cells expressing hTlR2/hTlR3 and for cells expressing only hTlR3 (J19-22). The fluorescence results obtained indicate that responses to these taste stimuli only occurred in the cells expressing hTlR2/hTlR3. Figure 6 contains 20 normalized dose-response curves, the results of which show that hTlR2 and hTlR3 function together as a human taste receptor based on their dose-specific interaction with various sweet stimuli. Particularly, Figure 6 contains dose-responses for sucrose, tryptophan and various other commercially available sweeteners. These results indicate that T1R2/T1R3 is a human sweet taste receptor as the rank order and threshold values 25 obtained in the assay closely mirror values for human sweet taste. 132

Example 8

Ligand-binding residues of mGluRl are conserved in T1R1

As shown in Figure 6, the key ligand-binding residues of mGluRl are conserved in T1R1. The interaction of glutamate with mGluRl is shown with several key residues highlighted according to the same color scheme as Figure 1.

Example 9

Human T1R1/T1R3 functions as umami taste receptors HEK cells stably expressing Gal 5 were transiently transfected with human T1R1, T1R3 and T1R1/T1R3 and assayed for increases in intracellular calcium in response to increasing concentrations of glutamate (Figure 8(a)), and 0.5 mM glutamate), 0.2 mM IMP, and 0.5 mM glutamate plus 0.2 mM IMP (Figure 8(b)). Human T1R1/T1R3 dose responses were determined for glutamate in the presence and absence of 0.2 mM IMP (Figure 8(c)). The maximal percentages of responding cells was approximately 5% for glutamate and approximately 10% for glutamate plus IMP. For clarity, does responses are normalized to the maximal percentage of responding cells. The values represent the mean + s.e. of four independent responses. X-axis circles mark taste detection thresholds determined by taste testing.

Example 10 PDZIP as an Export Sequence

The six residue PDZIP sequence (SVSTW (SEQ ID NO: 1)) was fused to the C-terminus of hTlR2 and the chimeric receptor (i.e. hTlR2-PDZIP) was transfected into an HEK-293 host cell. The surface expression of hTlR2 was then monitored using immunofluorescence and FACS scanning data. As shown in Figures 9A and 9B, the inclusion of the PDZIP sequence increased the surface expression of hTlR2-PDZIP relative to hTlR2. More specifically, Figure 9A shows an immunofluorescence staining of myc-tagged hT!R2 demonstrating that PDZIP significantly increases the amount of hTlR2 protein on the plasma membrane. Figure 9B shows FACS analysis data demonstrating the same result- Cells expressing myc-tagged hTlR2 are indicated by the dotted line and cells expressing myc-tagged hTlR2-PDZIP are indicated by the solid line. Particularly, Figure 10A shows untransfected Gal 5 stable host cells in HBS 133 1

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buffer, Figure 10B shows hTlR2-PDZIF transfected Gal5 stable hose cells in sweetener pool no. 5 (saccharin, sodium cyclamate, Acesulfame K, and Aspartame-20 mM each in HBS buffer), Figure IOC shows T1R3-PDZIP transfected Gal5 stable host cells in sweetener pool no. 5, and Figure 10D shows hTlR2-PDZIP/hTlR3-PDZlP co- 5 transfected Gal5 stable host cells in sweetener pool no. 5. Further, Figures 10E-10H show dose-dependent response of hTlR2/hTlR3 co-transfected Gal5 stable host cells to sucrose-E: OmM in HBS buffer; F: 30 mM; G: 60 mM; and H: 250 mM. Figures 101-1 OL shown the responses of hTlR2/hTlR3 co-transfected Gal5 stable host cells to individual sweeteners -1: Aspartame (1.5 mM); J: Acesulfame K (1 mM); K: Neotame 10 (20mM); L: Sodium cyclamate (20mM). As demonstrated by the calcium-images of

Figure 10, hTlR2 and hTlR3 are both required for the activities triggered by the sweet stimuli.

Example 11 15 Generation of Cell Lines that Stably Co-Express T1R1/T1R3 or T1R2/T1R3 Human cell lines that stably co-express human T1R2/T1R3 or human T1R1/T1R3 were generated by transfecting linearized PEAKlO-derived (Edge Biosystems) vectors and pCDNA 3.1/ZEO-derived (Invitrogen) vectors respectively containing hTIRl or hTlR2 expression construct (plasmid SAV2485 for T1R1, 20 SAV2486 for T1R2) and hTlR3 (plasmid SXV550 for T1R3) into a Gai5 expressing cell line. Specifically, T1R2/T1R3 stable cell lines were produced by co-transfecting linearized SAV2486 and SXV550 into Aurora Bioscience’s HEK-293 cell line that stably expresses Gai5· T1R1/T1R3 stable cell lines were produced by co-transfecting linearized SAV2485 and SXV550 into the same HEK-293 cell line that stably 25 expresses Gai5. Following SAV2485/SXV550 and SAV2486/SXV550 transfections, puromycin-resistant and zeocin-resistant colonies were selected, expanded, and tested by calcium imaging for responses to sweet or umami taste stimuli. Cells were selected in 0,0005 mg/ml puromycin (CALBIOCHEM) and 0.1 mg/ml zeocin (Invitrogen) at 37°C in low-glucose DMEM supplemented with GlutaMAX, 10% dialyzed FBS, and 30 0.003 mg/ml blasticidin. Resistant colonies were expanded, and their responses to sweet taste stimuli evaluated by Fluorescence microscopy. For automated fluorimetric imaging on VIPR-II instrumentation (Aurora Biosciences), T1R2/T1R3 stable cells were first seeded onto 96-well plates (approximately 100,000 cells per well). Twenty- 134

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four hours later, cells were loaded with the calcium dye fluo-3-AM (Molecular Probes), 0.005 mM in PBS, for one hour at room temperature. After replacement with 70 μΐ PBS, stimulation was performed at room temperature by addition of 70 μΐ PBS supplemented with taste stimuli. Fluorescence (480 nm excitation and 535 nm 5 emission) responses from 20 to 30 seconds following compound addition were averaged, corrected for background fluorescence measured prior to compound addition, and normalized to the response to 0.001 mM ionomycin (CALBIOCHEM), a calcium ionophore.

It was then observed that when these cell lines were exposed to sweet or umami 10 stimuli, that for active clones typically 80-100% of cells responded to taste stimuli.

Unexpectedly, the magnitude of individual cell responses was markedly larger than that of transiently transfected cells.

Based on this observation, the inventors tested the activity of T1R stable cell lines by automated fluorescence imaging using Aurora Bioscience’s VIPR 15 instrumentation as described above. The responses of two T1R1/T1R3 and one T1R2/T1R3 cell line are shown in Figure 11 and Figure 12 respectively.

Remarkably, the combination of increased numbers of responding cells and increased response magnitudes resulted in a greater than 10-fold increase in activity relative to transiently transfected cells. (By way of comparison, the percent ionomycin 20 response for cells transiently transfected with T1R2/T1R3 was approximately 5% under optimal conditions.) Moreover, dose responses obtained for stably expressed human T1R2/T1R3 and T1R1/T1R3 correlated with human taste detection thresholds. The robust T1R activity of these stable cell lines suggests that they are well suited for use in high-throughput screening of chemical libraries in order to identify compounds, e.g. 25 small molecules, that modulate the sweet or umami taste receptor and which therefore modulate, enhance, block or mimic sweet or umami taste.

Example 12

Generation of cell lines that inducibly co-express T1R1/T1R3 which selectively 30 respond to umami taste stimuli T1R1/T1R3 HEK 293 cell lines that stably expressed the umami taste receptor display robust improved activity relative to transiently tranfected cites. However, a disadvantage is that they can rapidly lose activity during cell propagation. 135

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Also, these findings show that (i) T1R1/T1R3 is a umami taste receptor, i.e., and (ii) that cell lines which robustly express T1R1/T1R3, preferably stable and/or inducible T1R1/T1R3 cell lines can be used in assays, preferably for high throughput screening of chemical libraries to identify novel modulators of umami taste. 5 Modulators that enhance umami taste may be used.

To overcome the instability of the T1R1/T1R3 stable cell lines, the HEK-Gai5 cells have been engineered to inducibly express T1R1/T1R3 using the GeneSwitch system (Invitrogen). pGene-derived zeocin-resistant expression vectors for human T1R1 and T1R3 (plasmid SXV603 for T1R1 and SXV611 for T1R3) and a puromycin- 10 resistant pSwitch-derived vector that carries the GeneSwitch protein (plasmid SXV628) were linearized and cotransfected into the HEK-Gais cell line. Zeocin-resistant and puromycin-resistant colonies were selected, expanded, induced with variable amounts of mifepristone, and tested by calcium imaging for responses to umami taste stimuli.

Inducible expression of T1R1/T1R3 resulted in robust activity. For example, 15 approximately 80% of induced cells but only approximately 10% of transiently transfected cells responded to L-glutamate; More specifically, pGene derived Zeocin-resistant expression vectors that express human T1R1 and human T1R3 and a puromycn-resistant pSwitch-derived vector that carries the GeneSwitch protein were linearized and co-tranfected into Gais cells. Cells were selected in 0.5 pg/ml 20 puromycin (CAL BIOCHEM) and 100 pg/ml Zeocin (Invitrogen) at 37°C in

Dulbecco’s Modified Eagle Medium supplemented with GlutaMAX, (10 % dialyzed FBS, and 3 ug/ml blasticidin. Resistant colonies were expanded, and their responses to umami taste stimuli following induction with 10'1θΜ mifepristone determined by fluorescence microscopy following the methods of Li et al., PNAS 99(7): 4692-4696 25 (2002).

For automated fluorometric imaging on FL1PR instrumentation (Molecular Device), cells from one clone (designated clone 1-17) were seeded into 96-well plates (approximately 80,000 cell per well) in the presence of 10'10 M mifepristone and incubated for 48 hours. Cells were then loaded with the calcium dye fluo-4-AM 30 (Molecular Probes), 3 μΜ in PBS, for 1.5 hours at room temperature.

After replacement with 50 μΐ PBS, stimulation was performed at room temperature by the addition of 50 μΐ PBS supplemented with different stimuli. In contrast to previous transient T1R1/T1R3 umami receptor expression systems that 136

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necessitated quantifying T1R1/T1R3 receptor activity by individually counting responding cells (Li et al., PNAS 99(7): 4692-4696 (2002)) (because of the low activity of the receptor therein), the subject inducible expression system resulted in a clone 1-17 having substantially increased activity that allowed receptor activity to be quantified by 5 determining maximal fluorescence increases (480 nm excitation and 535 nm emission) summated over fields of imaged cells. The maximal fluorescence from four independent determinations was averaged, corrected for background fluorescence measured prior to compound addition, and normalized to the response to 0.002 mM ionomycin (CALBIOCHEM). 10 These results are contained in Figure 13. Particularly, Figure 13 contains a dose-response curve determined for L-glutamate in the presence and absence of 0.2 mM IMP. In the figure, each value represents average summated maximal fluorescence (corrected for background fluorescence) for four independent determinations. These dose-response curves correspond to those determined for cells transiently transfected 15 with T1R1/TIR3.

The selectivity of the umami T1R1/T1R3 taste receptor was also evaluated by screening with different L-amino acids. The results obtained indicated that T1R1/T1R3 is selectively activated by the umami-tasting L-amino acids (L-glutamate and L-aspartate). 20 The results of experiments wherein the responses of the 1-17 clone was resulted in tested in the presence of different L-amino acids are contained in Figure 14 and Figure 15. Figure 14 shows the results of an experiment wherein the 1-17 cell line was contacted with different L-amino acids at a concentration of lOmM in the presence and absence of 1 mM IMP. 25 Figure 15 contains a dose-response curve for active amino acids determined in the presence of 0.2mM IMP. Each value represents the average of four independent determinations.

The results obtained in these experiments support the specificity and selectivity of the umami taste receptor to umami taste stimuli. Whereas the umami taste stimuli L- 30 glutamate and L-aspartate significantly activated the T1R1/T1R3 receptor at different concentrations (see Figure 14 and 15), the other L-amino acids which activated the human T1R1/T1R3 receptor only activated the receptor weakly and at much higher concentrations. 137

Therefore, these results support the selectivity of the T1R1/T1R3 receptor for umami taste stimuli and the suitability of this inducible stable expression system for use in high throughput screening assays using automated fluorometric imaging instrumentation to identify compounds that activate the umami taste receptor, for example L-glutamate or L-aspartate, or which enhance the activity of L-glutamate to activate the umami taste receptor, for example 5’-IMP or 5’-GMP, or block the activation of the umami taste receptor by umami taste stimuli such as L-glutamate and L-aspartate.

Compounds identified using these assays have potential application as flavorants in foods and beverage compositions for mimicing or blocking umami taste stimuli.

Example 13

Lactisole Inhibits the Receptor Activities of Human T1R2/T1R3 and T1R1/T1R3 , and Sweet and Umami Taste

Lactisole, an aralkyl carboxylic acid, was thought to be a selective sweet-taste inhibitor (See e.g., Lindley (1986) U.S. Patent 4,567,053; and Schiffman et al, Chem Senses 24:439-447 (1999)). Responses of HEK-Gai5 cells transiently transfected with T1R2/T1R3 to 150 mM sucrose in the presence of variable concentrations of lactisole were measured. Lactisole inhibits the activity of human T1R2/T1R3 with an IC50 of 24 μΜ.

The T1R1/T1R3 umami and T1R2/T1R3 sweet taste receptor may share a common subunit. It has therefore been theorized that lactisole, which inhibit the T1R2/T1R3 sweet taste receptor, may have a similar effect on the T1R1/T1R3 umami taste receptor. The present inventors tested the effect of lactisole on the response of human T1R1/T1R3 to lOmM L-Glutamate. As with the T1R2/T1R3 sweet receptor, lactisole inhibited T1R1/T1R3 with an IC50 of 165 μΜ. Lactisole inhibition likely reflects antagonism at the T1R receptors instead of, for example, non-specific inhibition of Gai5-mediated signaling because the response of muscarinic acetylcholine receptors was not inhibited by lactisole.

The present inventors then evaluated the effect of lactisole on human umami taste. Taste thresholds in the presence of 1 and 2 mM lactisole were determined for the umami taste stimuli L-Glutamate with or without 0.2 mM IMP, the sweet taste stimuli 138

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sucrose and D-tryptophan, and the salty taste stimulus sodium chloride following the methods of Schiffman et al. (Chem. Senses 24: 439-447 (1989)). Millimolar concentrations of lactisole dramatically increased detection thresholds for sweet and umami but not salt taste stimuli. These results are contained in Figure 16. 5 In conclusion, (i) these findings further support the inventors’ hypothesis that T1R1/T1R3 is the only umami taste receptor, and (ii) the T1R1/T1R3 and T1R2/T1R3 receptors may share a structurally related lactisole-binding domain.

While the foregoing detailed description has described several embodiments of the present invention, it is to be understood that the above description is illustrative 10 only and not limiting of the disclosed invention. The invention is to be limited only by the claims which follow.

Example 14

Mapping of Ligand Interaction Sites on the Sweet Receptor 15

Through coexpression of T1R2R-H with human T1R3, part of the human sweet receptor (the N-terminal domain of T1R2) was replaced with rat protein sequence. The responses to aspartame and neotame are abolished, showing that the N- terminal domain of human T1R2 is required for recognizing aspartame and neotame. Similarly, 20 the rat T1R2 N-terminal domain was also replaced with human protein sequence by coexpressing T1R2H-R with rat T1R3. The chimeric receptor gains the ability to respond to aspartame and neotame, suggesting that the same domain of human T1R2 is also sufficient (in the context of sweet receptors) to recognize those two sweeteners (Fig. 22B). These in vitro functional expression data indicate that the important 25 interaction determinants are located in the N-terminal extracellular domain.

In contrast, replacing either half of human T1R2 with rat protein sequence does not affect its response to cyclamate. Instead, the C-terminal domain of human T1R3 is required and sufficient, when co-expressed with T1R2, to recognize cyclamate (Fig. 22C). The transmembrane domain of family C GPCRs has been known to 30 contain binding sites for allosteric modulators (Gasparini, F., R. Kuhn, and J.P. Pin, Curr Opin Pharmacol 2002 Feb;2(1 ):43-9). This is the first case in family C GPCR, where an agonist binds directly to the transmembrane domain and activates the receptor in the absence of other ligand. . 139

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Lactisole, an aralkyl carboyxlic acid, is a specific human sweet taste inhibitor, which has physiological effect on the rodent taste. Consistent with the taste effect, lactisole inhibits the human but not rat T1R2/T1R3 response to sucrose in our assay system (Fig. 22A). The same kind of mapping experiments on lactisol

5 interaction site using the T1R chimeras was performed. Like cyclamate, lactisole requires the human T1R3 C-terminal domain to inhibit the receptor’s response to sucrose and acesulfame K (Fig. 22D). This result further demonstrates the importance of T1R3 C-terminal domain in the sweet receptor function. The chimeras in all 16 possible combinations 10 were tested, and all functional combinations generated results consistent with our model.

Mutagenesis studies were conducted on both T1R2 and T1R3 to narrow down the essential amino acids in recognition of aspartame, neotame, and cyclamate. If T1R2 and T1R3 are responsible for recognizing different sweeteners, mutations in 15 T1R2 N-terminal domain would affect responses to aspartame and neotame, but not cyclamate. In addition, mutations in T1R3 C-terminal domain would have the opposite effect. To select the crucial amino acid residues in the T1R2 N-terminal domain, the sequence of T1R2 was aligned with mGluRl (Fig. 23A). Among the eight residues that are crucial in ligand binding in mGluRl (Kunishima, N., et al., Nature, 2000. 20 407(6807): p. 971-7), three are conserved in human T1R2 (S144, Y218, and E302).

Each of the three residues were mutated and the resulting receptors were tested for their response to different sweeteners. Substitution of Y218 to A abolished the responses to all sweeteners tested, showing Y218 is important for the overall conformation of the receptor. The two other hTlR2 variants, containing SI 44 A and 25 E302A, selectively affected the response to aspartame and neotame but not cyclamate.

Stable cell lines expressing SI44A and E302A hTlR2 variants (coexpressed with wild type hTlR3 and Gais) did not respond to aspartame or neotame at the physiological concentrations, but did respond to cyclamate (Fig. 23B).

In order to further map the cyclamate-binding site, the three extracellular loops 30 in the T1R3 C-terminal domain were focused on. Alignment of human and rodent TlR3s reveal multiple amino acid differences in the three extracellular loops (Fig. 23C). Replacing extracellular loop-2 or loop-3 with rat sequences abolished the cyclamate response without affecting the sucrose or aspartame responses. In contrast, 140

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replacing extracellular loop 1 had no obvious effect on response to cyclamate, showing an important role for EC loops 2 and 3 in recognizing cyclamate (Fig. 23D). None of those loop-replacements affected the inhibition effect of lactisole, showing a different binding mechanism. In summary, amino acid substitutions in T1R2 or T1R3 result in 5 selective interference of activities induced by different sweeteners, consistent with the chimeric receptor results.

The above results demonstrate that the human sweet receptor function as a heteromeric complex of T1R2 and T1R3. Both subunits are required for recognizing different sweeteners, and the data indicate the existence of multiple binding pockets 10 on the receptor for different classes of agonists. The presence of multiple ligandbinding sites provides structural guidance and definition for the specifically binding compounds of the invention.

Example 15

Mapping of Receptor-G protein Interactions 15 The human and rat sweet receptors are also different in their G protein-coupling efficiency. Even though both human and rat receptors can couple efficiently to Gais/ii, only the human receptor can couple efficiently to Gtti5 (Fig. 24A). This species difference allows for mapping of the receptor G protein interactions using the same chimeric receptors as described above. T1R2 but not T1R3 appears to be critical for 20 Guts-coupling, since replacing the C-terminus of human T1R2 with the corresponding rat sequence abolished coupling, and replacing rat T1R2 C-terminal half with human sequence enabled the receptor to couple to Gtti5 and respond to sucrose and acesulfame K (Fig. 24); Swapping the T1R3 C-terminal sequences had no effect on Gtti5-coupling (Fig. 24B). This observation demonstrates the important role of T1R2 in G protein- 25 coupling in the functional expression system. Gustducin (Wong, G.T., K.S. Gannon, and R.F. Margolskee, Nature, 1996. 381(6585): p. 796-800) has been proposed to be an endogenous G protein for the sweet taste receptor, and T1R2 can be the subunit responsible for in vivo coupling in taste cells, GABAbR is the other example of heteromeric family C GPCR, whereas one subunit (GABAbRI) is responsible for 30 ligand- binding, and the other (GABAbR2) for G protein coupling (Margeta-Mitrovic, M., Faroe Natl Acad Sci U S A, 2001. 98(25): p. 14643-8; Margeta-Mitrovic, M., Proc Natl Acad Sci U S A, 2001. 98(25): p. 14649-54). The sweet receptor is different from GABAbR in that T1R2 is required for both ligand recognition and G- protein coupling. 141

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Example 16

Lactisole Antagonizes Human T1R1/T1R3 and Inhibits Human Umami

Taste 5 It was hypothesized that since T1R1/T1R3 function as heteromeric receptors as well as the sweet receptor, that lactisole should have similar effect on T1R1/T1R3 activity, since T1R3 is a common subunit between the sweet and the umami receptors. Indeed, lactisole antagonized human T1R1/T1R3 (Fig. 25A). Lactisole acts as a noncompetitive inhibitor of T1R1/T1R3, since the IC50 values are apparently not 10 dependent on glutamate concentration (Fig. 25B), and lactisole reduces the maximal activities of the receptor without significantly changing the ECso of agonists (Fig. 25C), These results demonstrate that lactisole binds to a different site from L-glutamate, and are consistent with the hypothesis that the glutamate-binding pocket is located in T1R1. Lactisole appears to be a competitive inhibitor of the sweet receptor, 15 as its IC50S are dependent on the concentrations of the sweeteners, and it increases the ECsos of the sweeteners without significantly affecting the maximal activities.

The inhibition effect of lactisole is mediated by the TIR receptors since it had no effect on the endogenous muscarinic acetylcholine receptor in HEK cells or on a mouse bitter receptor, mT2R5, transiently expressed in HEK cells. As was the case 20 for the T1R2/T1R3 receptor, lactisole inhibition of the T1R1/T1R3 response to umami taste stimuli was reversible following washout and restimulation.

To correlate the receptor activity with behavior, the effect of lactisole on human umami taste was tested. As predicted, millimolar concentrations of lactisole dramatically increased detection thresholds for sweet and umami but not salt taste 25 stimuli (Fig. 25D). Lactisole was previously not known to be an umami taste inhibitor. The correlation between receptor activity and taste results demonstrates a crucial role of TIRs in human umami taste.

Example 17

Cyclamate Enhances Human T1R1/T1R3 Receptor Activities 30 Based on the same heteromeric model of TIRs (Fig. 26), it was predicted that cyclamate would also modulate the activity of the human T1R1/T1R3 umami receptor by acting on T1R3. Although cyclamate alone had no effect on T1R1/T1R3, it enhanced the activity of the receptor in the presence of L-glutamate (Fig. 27E). This 142

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effect is specific for the human T1R1/T1R3, as cyclamate had no effect on the activities of the endogenous muscarinic acetylcholine receptor in the presence of carbachol (Fig. 27E). It is noteworthy that cyclamate has comparable EC50S for the sweet receptor (Fig. 23B) and umami receptor. Cyclamate reproducibly left-shifts the 5 dose-response curves for L-glutamate by -2 fold in the presence or absence of IMP (Fig. 25F). IMP has a more dramatic effect of enhancing the receptor, and the effect of cyclamate is observed in the presence of IMP (Fig. 25F), suggesting a different mechanism from IMP in enhancing the receptor. IMP appears to bind to T1R1, since it has no effect on the 10 sweet receptor. Other sweeteners, including sucrose, aspartame, saccharin, and D-tryptophan, had no effect on the human T1R1/T1R3 activities.

In summary, it has been demonstrated that both T1R2 and T1R3 are required in a functional sweet receptor, that aspartame and neotame require the N-terminal extracellular domain of T1R2, G protein-coupling requires C-terminal half of T1R2, 15 and that cyclamate and lactisole require the transmembrane domain of T1R3. These findings demonstrate the different functional roles ofTIR subunits in a heteromeric complex and the presence of multiple sweetener interaction sites on the sweet receptor. Because T1R3 is the common subunit in the sweet and the umami receptors, it was predicted and confirmed the effect of cyclamate and lactisole on the umami 20 receptor. Furthermore, a correlation was able to be made between the lactisole effect on the receptor activities with taste. Based on these observations, a model was created (Fig. 26) for the structure-function relationships of the T1R family taste receptors. Natural carbohydrate sweeteners bind to the N-terminal domain of T1R2, similar to aspartame and neotame, and there are other ligand binding sites on the sweet receptor 25 as well, for example, the transmembrane domain of T1R2. The umami receptor functions similarly as a heteromeric complex, and MSG and IMP each appears to bind to the T1R1 subunit, since neither has any effect on the sweet receptor, and the transmembrane domain of T1R1 is responsible for coupling to G proteins. 143

Example 18 HTS Protocol for Sweet Tastants

An HEK293 cell line derivative (Chandrashekar, J., Mueller, K.L., Hoon, M.A., Adler, E., Feng, L., Guo, W., Zuker, C.S, Ryba, N.J.,. Cel,12000,100, 703-711.) that stably expresses Gal5 and hTlR2/hTlR3 (Li, X., Staszewski, L., Xu, H., Durick, K., Zoller, M., Adler, E. Proc Natl Acad Set US A 2002, 99, 4692-4696, World Patent # WO 03/001876 A2, herein incorporated by reference in their entirety) was used to identify compounds with sweet taste enhancing properties.

Compounds were initially selected based on their activity on the hTlR2/hTlR3-HEK293-Gal5 cell line (Li, et al. vide supra). Activity was determined using an automated fluorometric imaging assay on a FLIPR instrument (Fluorometric Intensity Plate Reader, Molecular Devices, Sunnyvale, CA) (designated FLIPR assay). Cells from one clone (designated S-9 cells) were seeded into 384-well plates (at approximately 50,000 cells per well) in a medium containing DMEM Low Glucose (Invitrogen, Carlsbad, CA), 10% dialyzed fetal bovine serum (Invitrogen, Carlsbad, CA), 100 Units/ml Penicillin G, and 100 pg/ml Streptomycin (Invitrogen, Carlsbad, CA) (Li, et al. vide supra) see also World Patent #WO 03/001876 A2). S-9 cells were grown for 24 hours at 37 °C. S-9 cells were then loaded with the calcium dye Fluo-3 AM (Molecular Probes, Eugene, OR), 4 μΜ in a phosphate buffered saline (D-PBS) (Invitrogen, Carlsbad, CA), for 1 hour at room temperature. After replacement with 25 μΐ D-PBS, stimulation was performed in the FLIPR instrument and at room temperature by the addition of 25 μΐ D-PBS supplemented with different stimuli at concentrations corresponding to twice the desired final level. Receptor activity was quantified by determining the maximal fluorescence increases (using a 480 nm excitation and 535 nm emission) after normalization to basal fluorescence intensity measured before stimulation.

For dose-responses analysis, stimuli were presented in duplicates at 10 different concentrations ranging from 60 nM to 30 μΜ. Activities were normalized to the response obtained with 400 mM D-fructose, a concentration that elicits maximum receptor response, EC50S were determined using a non-linear regression algorithm 144

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(using Senomyx, Inc. software), where the Hill slope, bottom asymptotes and top asymptotes were allow to vary. Identical results were obtained when analyzing the dose-response data using commercially available software for non-linear regression analysis such as GraphPad PRISM (San Diego, CA). 5 In order to determine the dependency of hTlR2/hTlR3 for the cell response to different stimuli, selected compounds were subjected to a similar analysis on HEK293-Gal5 cells (not expressing the human sweet receptor). The HEK293-Gal5 cells do not show any functional response in the FLIPR assay to D-Fructose or any other known sweeteners. Similarly, compounds described herein do not induce any functional 10 response when using HEK293-Gal5 cells in the FLIPR assay.

Example 19

Flavor Enhancement Measurements for Sweet Tastants using Human Volunteers

Basic screening of sensory taste testers: Potential panelists were tested for their abilities to rank and rate intensities of solutions representing the five basic tastes. 15 Panelists ranked and rated intensity of five different concentrations of each of the five following compounds: sucrose (sweet), sodium chloride (salty), citric acid (sour), caffeine (bitter), and monosodium glutamate (umami). Panelists tasted a total of 25 samples per session (5 samples of each of the 5 solution types). In the first session, panelists ranked the five concentrations for intensity of the attribute in question. This 20 was repeated four more times with other samples. In the second session, panelists rated intensity of the five concentrations of each sample using a line scale called the “Labeled Magnitude Scale” (LMS). The LMS is anchored with intensities (e.g. barely detectable, weak, moderate, strong, very strong, and strongest imaginable) to assist panelists in rating the samples. Samples were tasted in 10ml volumes at room 25 temperature and labeled with 3-digit blinding codes. Samples were presented in randomized, counterbalanced order within each sample solution (e.g. sucrose, citric acid, etc.).

In order to be selected for participation in testing, panelists needed to correctly rank and rate samples for intensity, with a reasonable number of errors. Approximately 30 25 people successfully completed this procedure.

Panelists selected in the above procedure were deemed qualified for performing

Preliminary Taste Testing procedures. The preliminary taste tests are used to evaluate 145

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new compounds for intensity of basic tastes and off-tastes. A small group of panelists (n=5) taste approximately 5 concentrations of the compound (range typically between Ι-lOOuM, in half-log cycles, e.g. 1,3, 10, 30, and lOOuM) in water or buffer and in a solution of 4% (w/v, 117 mM) sucrose to evaluate enhancement. Typically samples 5 also contain 0.1% ethanol in order to aid dispersion of the compound in a water-based solution. Panelists rate the five basic tastes (sweet, salty, sour, bitter, and umami) as well as off-tastes (such as chemical, metallic, sulfur) on the LMS. Samples are served in 10ml portions at room temperature. The purpose of the test is to determine the highest concentration at which there is no objectionable off-taste, and determine if 10 obvious enhancement of sweet taste exists at any of the concentrations tested.

If the compound is effective and does not have objectionable off-tastes, it is tested with a trained (expert panel) in a larger study.

For example: Five panelists evaluated 1, 3, 10, 30, and lOOuM XVI-3 in water and in 4% sucrose solution. All samples with compound were balanced for ethanol at 15 0.1% (aids in dispersion of compound). Panelists were asked to rate basic tastes and off-tastes using the LMS for each sample tasted. When panelists noted sweetness in any sample, they were asked to taste reference samples of sucrose (2, 4, 6, 8% sucrose) to estimate equivalent sweetness. A trained (expert) panel was used to further evaluate compounds that had been 20 tested with the preliminary taste test.

Panelists for the trained panel were selected from the larger group of qualifying taste panelists. Panelists were further trained on sweet taste by ranking and rating experiments using sucrose solutions. Panelists completed a series of ranking, rating, and difference from reference tests with sweet solutions. In ranking and rating 25 experiments, panelists evaluated sucrose concentrations (2, 4, 6, 8 % (w/v)) sucrose.

Compounds tested by the trained panel were evaluated in difference from reference experiments. Panelists were given reference samples of various concentrations (2,4,6, or 8 % (w/v) sucrose) and asked to rate samples on a scale of -5 to +5 in terms of difference in sweet taste from the reference (score: -5= much less 30 sweet taste than the reference; 0=same sweet taste as the reference; +5=much more sweet taste than the reference). Test samples were solutions with varying amounts of sucrose and compound. Typically, each session compared the reference sample (labeled as REF) to numerous test samples (labeled with 3-digit blinding codes). Tests 146

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typically included various samples with varying concentrations of sucrose, as well as one blind sample of the reference itself, to evaluate panel accuracy. Compounds were tested against the reference in samples with and without 4% or 6% sucrose. All samples were presented in 10ml volumes at room temperature. Futhermore, to 5 determine the sweetness of the compound alone, a reference solution was prepared at the designated concentration and compared to the threshold sweetness of sucrose (2%).

Example 20 HTS Protocol for Umami Tastants HEK-Gai5 cells were engineered to inducibly express T1R1/T1R3 using the 10 GeneSwitch system (Invitrogen). pGene-derived zeocin-resistant expression vectors for human T1R1 and T1R3 (plasmid SXV603 for T1R1 and SXV611 for T1R3) and a puromycin-resistant pSwitch-derived vector that carries the GeneSwitch protein (plasmid SXV628) were linearized and cotransfected into the HEK-Gai5 cell line. Zeocin-resistant and puromycin-resistant colonies were selected, expanded, induced 15 with variable amounts of mifepristone, and tested by calcium imaging for responses to umami taste stimuli. Cells were selected in 0.5 pg/ml puromycin (CAL BIOCHEM) and 100 pg/ml Zeocin (Invitrogen) at 37°C in Dulbecco’s Modified Eagle Medium supplemented with GlutaMAX, (10 % dialyzed FBS, and 3 ug/ml blasticidin. Resistant colonies were expanded, and their responses to umami taste stimuli following induction 20 with 10'10 M mifepristone determined by fluorescence microscopy following the methods of Li, et ai, PNAS (2002) 99(7):4692-4696. For automated fluorometric imaging on FLIPR instrumentation (Molecular Device), cells from one clone (designated clone 1-17) were seeded into 96- or 384-well plates (approximately 80,000 cell per well) in the presence of IO'10 M mifepristone and incubated for 48 hours. Cells 25 were then loaded with the calcium dye fluo-4-AM (Molecular Probes), 3 pM in PBS, for 1.5 hours at room temperature. After replacement with 50 pi PBS, stimulation was performed at room temperature by the addition of 50 pi PBS supplemented with different stimuli. The maximal fluorescence from four independent determinations were averaged, corrected for background fluorescence measured prior to compound 30 addition, and normalized to the response to 0.002 mM ionomycin (CALBIOCHEM). 147

Example 21

Taste Test Protocol for Umami Tastants

Basic Training of Sensory Tasters: Tasters were trained to evaluate the taste of aqueous solutions (5 mL each, “swash and spit”) of the following standard taste compounds by using the triangle test as described in the literature: sucrose (50 mM) for sweet taste; citric acid (5 mM) or lactic acid (20 mM) for sour taste; NaCl (12 mM) for salty taste, quinine (10 μΜ) or caffeine (1 mM) for bitter taste; and monosodium glutamate (8 mM) for umami or “savory” taste.

Training for Umami Taste: Tasters were given 1-3 sets of 6 MSG and/or MSG-IMP samples ranging from 3-60 mM MSG and 0-200 μΜ IMP, each arranged in the tray in ascending concentration. This exercise gave the subject practice doing dose response evaluations. Then another set was made up of the same six samples, but were given in random order. The subject was then asked to arrange the samples in ascending intensity and then to rate their umami intensity.

Qualifying Taste Panelists: Tasters were subjected to a standard two alternative forced choice (2AFC) test with 5 pairs of taste samples. They were asked to make a choice of the most umami sample from two samples (a pair). The test contains two easy pairs, two with medium difficulty, and one difficult pair. Tasters who could differentiate the medium difficulty pairs were selected as panelists.

Pilot/Qualitative taste test of Umami Enhancer Candidate (UEC) by a small group of panelists: Taste samples of appropriate concentrations (usually 1-50 μΜ) were made in water (use minimum amount of ethanol if not soluble); Taste UEC alone at 30 and/or 50 μΜ for umami and other attributes. Rate those taste attributes on the appropriate Labeled Magnitude Scale (LMS) on the screening ballot; if UEC has no/low umami and other tastes, then move forward to discrimination test; compare certain concentration of MSG, e.g., 12 mM and 12 mM MSG + 30 μΜ UEC to determine if there is any enhancement; rate the perceived umami intensity on the appropriate LMS on the screening ballot; vary concentration of UEC and/or MSG to find the best combination; decide what solutions to use in panel screening; record all procedures and data including description of study, sample prep, sample arrangement, ballots and sign up sheet for panelists, data entry and evaluation. 148 2AFC Panel Screening of UEC: Run panel screening with qualified panelists using protocols generated from the pilot tasting; record all procedures and data; prepare summary report with statistically significant conclusions, if any.

Example 22

Quantitative Taste Tests for Compounds 2725761 and 3756807

Quantitative taste tests for compounds 2725761 and 3756807 were run according to procedures presented above. It was found that both of them have some enhancement for MSG, in addition to their additive effect of the umami intensity.

Example 23

Synthesis of Compounds 2725761 and 3756807

Compounds 2725761 and 3756807 are prepared as shown in Example 22, from their corresponding acids and amines. The products are purified by conventional methods, e.g., basic and acidic aqueous washes, or preparative HPLC. The structures of those compounds were confirmed based on usual analytical methods, e.g, NMR and LCMS. This method can also be used to synthesize any of the compounds found in Tables 1-5.

Example 24 Cell Based Assays

Cells were grown and maintained at 37°C in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and MEM non-essential amino acids (Gibco BRL); media for Gais cells also contained 3 gg ml*1 blasticidin (Gibco BRL). For calcium-imaging experiments, cells were first seeded onto 48-well tissue-culture plates (approximately 30,000 cells per well), and transfected using Mirus Transit-293 (PanVera). Transfection efficiencies, which were estimated by cotransfection with an RFP expression vector, were typically approximately 60%. To minimize glutamate-induced and glucose-induced desensitization, supplemented DMEM was replaced with low-glucose DMEM supplemented with GlutaMAX and 10% dialyzed FBS (Gibco BRL) approximately 24 hours after transfection. After an additional 24 hours, cells were loaded with the calcium dye fluo-4-AM (Molecular Probes), 3 μΜ in Dulbecco’s PBS buffer (DPBS, GibcoBRL), for 1.5 hours at room temperature. After replacement 149

<img img-format="tif" img-content="drawing" file="IL173552AD000290.tif" id="idf0090" />

with 100 μΐ DPBS, stimulation was performed at room temperature by addition of 100 μΐ DPBS supplemented with taste stimuli. Calcium mobilization was monitored on an Axiovert S100 microscope equipped with an inverted 10X/0.5 LWD piano fluor objective (Zeiss) and a cooled CCD camera (Princeton Instruments). Fluorescence 5 images were acquired at 480 nm excitation and 535 nm emission, and analyzed with Imaging Workbench 4.0 software (Axon Instruments). T1R receptor activity was quantitated by counting the number of responding cells 30 seconds after stimulus addition. 150 173,552/5

SEQUENCE LISTING &lt;110&gt; Senomyx, Inc. &lt;120&gt; TIR HETERO-OLIGOMERIC TASTE RECEPTORS,

CELL LINES THAT EXPRESS SAID RECEPTORS, AND TASTE COMPOUNDS &lt;130&gt; 19328.0001P1 &lt;140&gt; &lt;141&gt; PCT/US2004/025459 2004-08-06 &lt;150&gt; &lt;151&gt; 60/494,071 2003-08-06 &lt;150&gt; &lt;151&gt; 60/552,064 2004-03-09 &lt;160&gt; 33 &lt;170&gt; FastSEQ for Windows Version 4.0 &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt; 1 5 PRT Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 1

Ser Val Ser Thr Trp 1 5 &lt;210&gt; &lt;211&gt; &lt;212&gt; &lt;213&gt; 2 14 PRT Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;221&gt; &lt;222&gt; &lt;223&gt; VARIANT 1,6 Xaa = Thr or Arg

&lt;221&gt; VARIANT &lt;222&gt; 3 &lt;223&gt; Xaa = Phe or Leu

&lt;221&gt; VARIANT &lt;222&gt; 4 &lt;223&gt; Xaa = Arg, Gin, or Pro

&lt;221&gt; VARIANT 151 173,552/6 &lt;222&gt; &lt;223&gt; 7 Xaa = Ser, Pro or Val &lt;221&gt; &lt;222&gt; &lt;223&gt; VARIANT 8 Xaa = Val, Glu, Arg, Lys or Thr &lt;221&gt; &lt;222&gt; &lt;223&gt; VARIANT 11 Xaa = Ala or Glu &lt;221&gt; &lt;222&gt; &lt;223&gt; VARIANT 12 Xaa = Trp or Leu &lt;221&gt; &lt;222&gt; &lt;223&gt; VARIANT 13 Xaa = Arg, His or Gly &lt;400&gt; 2

Xaa Cys Xaa Xaa Arg Xaa Xaa Xaa Phe Leu Xaa Xaa Xaa Glu 1 5 10 &lt;210&gt; 3 &lt;211&gt; 15

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;221&gt; &lt;222&gt; &lt;223&gt; VARIANT 1 Xaa = Leu or Gin &lt;221&gt; &lt;222&gt; &lt;223&gt; VARIANT 3 Xaa = Glu, Gly or Thr &lt;221&gt; &lt;222&gt; &lt;223&gt; VARIANT 4 Xaa = Asn, Arg or Cys &lt;221&gt; &lt;222&gt; VARIANT 7 &lt;223&gt; Xaa = Arg or Glu

&lt;221&gt; VARIANT &lt;222&gt; 9 &lt;223&gt; Xaa = Arg or Lys

&lt;221&gt; VARIANT &lt;222&gt; 10 152 173,552/5 &lt;223&gt; Xaa = Cys , Gly or Phe &lt;221&gt; &lt;222&gt; &lt;223&gt; VARIANT 11 Xaa = Val , Leu or lie &lt;221&gt; &lt;222&gt; &lt;223&gt; VARIANT 13 Xaa = Phe or Leu &lt;221&gt; &lt;222&gt; &lt;223&gt; VARIANT 14 Xaa = Ala or Ser &lt;221&gt; VARIANT &lt;222&gt; 15 &lt;223&gt; Xaa = Met or Leu &lt;400&gt; 3 Xaa Pro Xaa Xaa Tyr Asn Xaa Ala Xaa Xaa Xaa Thr Xaa Xaa Xaa 1 5 10 15 &lt;210&gt; 4 &lt;211&gt; 858

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 4 Met Pro Gly Leu Ala lie Leu Gly Leu Ser Leu Ala Ala Phe Leu Glu 1 5 10 15 Leu Gly Met Gly Ser Ser Leu Cys Leu Ser Gin Gin Phe Lys Ala Gin 20 25 30 Gly Asp Tyr He Leu Gly Gly Leu Phe Pro Leu Gly Thr Thr Glu Glu 35 40 45 Ala Thr Leu Asn Gin Arg Thr Gin Pro Asn Gly He Leu Cys Thr Arg 50 55 60 Phe Ser Pro Leu Gly Leu Phe Leu Ala Met Ala Met Lys Met Ala Val 65 70 75 80 Glu Glu lie Asn Asn Gly Ser Ala Leu Leu Pro Gly Leu Arg Leu Gly 85 90 95 Tyr Asp Leu Phe Asp Thr Cys Ser Glu Pro Val Val Thr Met Lys Pro 100 105 110 Ser Leu Met Phe Met Ala Lys Val Gly Ser Gin Ser He Ala Ala Tyr 115 120 125 Cys Asn Tyr Thr Gin Tyr Gin Pro Arg Val Leu Ala Val He Gly Pro 130 135 140 His Ser Ser Glu Leu Ala Leu He Thr Gly Lys Phe Phe Ser Phe Phe 145 150 155 160 Leu Met Pro Gin Val Ser Tyr Ser Ala Ser Met Asp Arg Leu Ser Asp 165 170 175 Arg Glu Thr Phe Pro Ser Phe Phe Arg Thr Val Pro Ser Asp Arg Val 180 185 190 Gin Leu Gin Ala Val Val Thr Leu Leu Gin Asn Phe Ser Trp Asn Trp 195 200 205 153 173,552/2

Val Ala 210 Ala Leu Gly Ser Asp Asp Asp Tyr Gly Arg Glu Gly Leu Ser 215 220 He Phe Ser Gly Leu Ala Asn Ser Arg Gly lie Cys lie Ala His Glu 225 230 235 240 Gly Leu Val Pro Gin His Asp Thr Ser Gly Gin Gin Leu Gly Lys Val 245 250 255 Val Asp Val Leu Arg Gin Val Asn Gin Ser Lys Val Gin Val Val Val 260 265 270 Leu Phe Ala Ser Ala Arg Ala Val Tyr Ser Leu Phe Ser Tyr Ser lie 275 280 285 Leu His Asp Leu Ser Pro Lys Val Trp Val Ala Ser Glu Ser Trp Leu 290 295 300 Thr Ser Asp Leu Val Met Thr Leu Pro Asn lie Ala Arg Val Gly Thr 305 310 315 320 Val Leu Gly Phe Leu Gin Arg Gly Ala Leu Leu Pro Glu Phe Ser His 325 330 335 Tyr Val Glu Thr Arg Leu Ala Leu Ala Ala Asp Pro Thr Phe Cys Ala 340 345 350 Ser Leu Lys Ala Glu Leu Asp Leu Glu Glu Arg Val Met Gly Pro Arg 355 360 365 Cys Ser Gin Cys Asp Tyr lie Met Leu Gin Asn Leu Ser Ser Gly Leu 370 375 380 Met Gin Asn Leu Ser Ala Gly Gin Leu His His Gin lie Phe Ala Thr 385 390 395 400 Tyr Ala Ala Val Tyr Ser Val Ala Gin Ala Leu His Asn Thr Leu Gin 405 410 415 Cys Asn Val Ser His Cys His Thr Ser Glu Pro Val Gin Pro Trp Gin 420 425 430 Leu Leu Glu Asn Met Tyr Asn Met Ser Phe Arg Ala Arg Asp Leu Thr 435 440 445 Leu Gin Phe Asp Ala Lys Gly Ser Val Asp Met Glu Tyr Asp Leu Lys 450 455 460 Met Trp Val Trp Gin Ser Pro Thr Pro Val Leu His Thr Val Gly Thr 465 470 475 480 Phe Asn Gly Thr Leu Gin Leu Gin His Ser Lys Met Tyr Trp Pro Gly 485 490 495 Asn Gin Val Pro Val Ser Gin Cys Ser Arg Gin Cys Lys Asp Gly Gin 500 505 510 Val Arg Arg Val Lys Gly Phe His Ser Cys Cys Tyr Asp Cys Val Asp 515 520 525 Cys Lys Ala Gly Ser Tyr Arg Lys His Pro Asp Asp Phe Thr Cys Thr 530 535 540 Pro Cys Gly Lys Asp Gin Trp Ser Pro Glu Lys Ser Thr Thr Cys Leu 545 550 555 560 Pro Arg Arg Pro Lys Phe Leu Ala Trp Gly Glu Pro Ala Val Leu Ser 565 570 575 Leu Leu Leu Leu Leu Cys Leu Val Leu Gly Leu Thr Leu Ala Ala Leu 580 585 590 Gly Leu Phe Val His Tyr Trp Asp Ser Pro Leu Val Gin Ala Ser Gly 595 600 605 Gly Ser Leu Phe Cys Phe Gly Leu He Cys Leu Gly Leu Phe Cys Leu 610 615 620 Ser Val Leu Leu Phe Pro Gly Arg Pro Arg Ser Ala Ser Cys Leu Ala 625 630 635 640 Gin Gin Pro Met Ala His Leu Pro Leu Thr Gly Cys Leu Ser Thr Leu 645 650 655 Phe Leu Gin Ala Ala Glu He Phe Val Glu Ser Glu Leu Pro Leu Ser 660 665 670 Trp Ala Asn Trp Leu Cys Ser Tyr Leu Arg Gly Pro Trp Ala Trp Leu 675 680 685 154 173,552/2

Val Val 690 Leu Leu Ala Thr Leu 695 Val Glu Ala Ala Leu 700 Cys Ala Trp Tyr Leu Met Ala Phe Pro Pro Glu Val Val Thr Asp Trp Gin Val Leu Pro 705 710 715 720 Thr Glu Val Leu Glu His Cys Arg Met Arg Ser Trp Val Ser Leu Gly 725 730 735 Leu Val His He Thr Asn Ala Val Leu Ala Phe Leu Cys Phe Leu Gly 740 745 750 Thr Phe Leu Val Gin Ser Gin Pro Gly Arg Tyr Asn Arg Ala Arg Gly 755 760 765 Leu Thr Phe Ala Met Leu Ala Tyr Phe lie He Trp Val Ser Phe Val 770 775 780 Pro Leu Leu Ala Asn Val Gin Val Ala Tyr Gin Pro Ala Val Gin Met 785 790 795 800 Gly Ala lie Leu Phe Cys Ala Leu Gly lie Leu Ala Thr Phe His Leu 805 810 815 Pro Lys Cys Tyr Val Leu Leu Trp Leu Pro Glu Leu Asn Thr Gin Glu 820 825 830 Phe Phe Leu Gly Arg Ser Pro Lys Glu Ala Ser Asp Gly Asn Ser Gly 835 840 845 Ser Ser Glu Ala Thr Arg Gly His Ser Glu 850 855 &lt;210&gt; 5 &lt;211&gt; 841

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 5

Met Leu Leu Cys Thr Ala Arg Leu Val Gly Leu Gin Leu Leu lie Ser 1 5 10 15 Cys Cys Trp Ala Phe Ala Cys His Ser Thr Glu Ser Ser Pro Asp Phe 20 25 30 Thr Leu Pro Gly Asp Tyr Leu Leu Ala Gly Leu Phe Pro Leu His Ser 35 40 45 Gly Cys Leu Gin Val Arg His Arg Pro Glu Val Thr Leu Cys Asp Arg 50 55 60 Ser Cys Ser Phe Asn Glu His Gly Tyr His Leu Phe Gin Ala Met Arg 65 70 75 80 Leu Gly Val Glu Glu He Asn Asn Ser Thr Ala Leu Leu Pro Asn He 85 90 95 Thr Leu Gly Tyr Gin Leu Tyr Asp Val Cys Ser Asp Ser Ala Asn Val 100 105 110 Tyr Ala Thr Leu Arg Val Leu Ser Leu Pro Gly Gin His His He Glu 115 120 125 Leu Gin Gly Asp Leu Leu His Tyr Ser Pro Thr Val Leu Ala Val He 130 135 140 Gly Pro Asp Ser Thr Asn Arg Ala Ala Thr Thr Ala Ala Leu Leu Ser 145 150 155 160 Pro Phe Leu Val Pro Met lie Ser Tyr Ala Ala Ser Ser Glu Thr Leu 165 170 175 Ser Val Lys Arg Gin Tyr Pro Ser Phe Leu Arg Thr lie Pro Asn Asp 180 185 190 Lys Tyr Gin Val Glu Thr Met Val Leu Leu Leu Gin Lys Phe Gly Trp 195 200 205 Thr Trp lie Ser Leu Val Gly Ser Ser Asp Asp Tyr Gly Gin Leu Gly 210 215 220 155 173,552/2

Val Gin Ala Leu Glu Asn Gin Ala Thr Gly Gin Gly Ile Cys Ile Ala 225 230 235 240 Phe Lys Asp Ile Met Pro Phe Ser Ala Gin Val Gly Asp Glu Arg Met 245 250 255 Gin Cys Leu Met Arg His Leu Ala Gin Ala Gly Ala Thr Val Val Val 260 265 270 Val Phe Ser Ser Arg Gin Leu Ala Arg Val Phe Phe Glu Ser Val Val 275 280 285 Leu Thr Asn Leu Thr Gly Lys Val Trp Val Ala Ser Glu Ala Trp Ala 290 295 300 Leu Ser Arg His Ile Thr Gly Val Pro Gly Ile Gin Arg Ile Gly Met 305 310 315 320 Val Leu Gly Val Ala Ile Gin Lys Arg Ala Val Pro Gly Leu Lys Ala 325 330 335 Phe Glu Glu Ala Tyr Ala Arg Ala Asp Lys Lys Ala Pro Arg Pro Cys 340 345 350 His Lys Gly Ser Trp Cys Ser Ser Asn Gin Leu Cys Arg Glu Cys Gin 355 360 365 Ala Phe Met Ala His Thr Met Pro Lys Leu Lys Ala Phe Ser Met Ser 370 375 380 Ser Ala Tyr Asn Ala Tyr Arg Ala Val Tyr Ala Val Ala His Gly Leu 385 390 395 400 His Gin Leu Leu Gly Cys Ala Ser Gly Ala Cys Ser Arg Gly Arg Val 405 410 415 Tyr Pro Trp Gin Leu Leu Glu Gin Ile His Lys Val His Phe Leu Leu 420 425 430 His Lys Asp Thr Val Ala Phe Asn Asp Asn Arg Asp Pro Leu Ser Ser 435 440 445 Tyr Asn Ile Ile Ala Trp Asp Trp Asn Gly Pro Lys Trp Thr Phe Thr 450 455 460 Val Leu Gly Ser Ser Thr Trp Ser Pro Val Gin Leu Asn Ile Asn Glu 465 470 475 480 Thr Lys Ile Gin Trp His Gly Lys Asp Asn Gin Val Pro Lys Ser Val 485 490 495 Cys Ser Ser Asp Cys Leu Glu Gly His Gin Arg Val Val Thr Gly Phe 500 505 510 His His Cys Cys Phe Glu Cys Val Pro Cys Gly Ala Gly Thr Phe Leu 515 520 525 Asn Lys Ser Asp Leu Tyr Arg Cys Gin Pro Cys Gly Lys Glu Glu Trp 530 535 540 Ala Pro Glu Gly Ser Gin Thr Cys Phe Pro Arg Thr Val Val Phe Leu 545 550 555 560 Ala Leu Arg Glu His Thr Ser Trp Val Leu Leu Ala Ala Asn Thr Leu 565 570 575 Leu Leu Leu Leu Leu Leu Gly Thr Ala Gly Leu Phe Ala Trp His Leu 580 585 590 Asp Thr Pro Val Val Arg Ser Ala Gly Gly Arg Leu Cys Phe Leu Met 595 600 605 Leu Gly Ser Leu Ala Ala Gly Ser Gly Ser Leu Tyr Gly Phe Phe Gly 610 615 620 Glu Pro Thr Arg Pro Ala Cys Leu Leu Arg Gin Ala Leu Phe Ala Leu 625 630 635 640 Gly Phe Thr lie Phe Leu Ser cys Leu Thr Val Arg Ser Phe Gin Leu 645 650 655 Ile Ile Ile Phe Lys Phe Ser Thr Lys Val Pro Thr Phe Tyr His Ala 660 665 670 Trp Val Gin Asn His Gly Ala Gly Leu Phe Val Met Ile Ser Ser Ala 675 680 685 Ala Gin Leu Leu Ile Cys Leu Thr Trp Leu Val Val Trp Thr Pro Leu 690 695 700 156 173,552/2

Pro 705 Ala Arg Glu Tyr Gin 710 Arg Phe Pro His Leu 715 Val Met Leu Glu Cys 720 Thr Glu Thr Asn Ser Leu Gly Phe He Leu Ala Phe Leu Tyr Asn Gly 725 730 735 Leu Leu Ser lie Ser Ala Phe Ala Cys Ser Tyr Leu Gly Lys Asp Leu 740 745 750 Pro Glu Asn Tyr Asn Glu Ala Lys Cys Val Thr Phe Ser Leu Leu Phe 755 760 765 Asn Phe Val Ser Trp lie Ala Phe Phe Thr Thr Ala Ser Val Tyr Asp 770 775 780 Gly Lys Tyr Leu Pro Ala Ala Asn Met Met Ala Gly Leu Ser Ser Leu 785 790 795 800 Ser Ser Gly Phe Gly Gly Tyr Phe Leu Pro Lys Cys Tyr Val lie Leu 805 810 815 Cys Arg Pro Asp Leu Asn Ser Thr Glu His Phe Gin Ala Ser He Gin 820 825 830 Asp Tyr Thr Arg Arg Cys Gly Ser Thr 835 840 &lt;210&gt; 6 &lt;211&gt; 839

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 6

Met Gly Pro Arg Ala Lys Thr lie Cys Ser Leu Phe Phe Leu Leu Trp 1 5 10 15 Val Leu Ala Glu Pro Ala Glu Asn Ser Asp Phe Tyr Leu Pro Gly Asp 20 25 30 Tyr Leu Leu Gly Gly Leu Phe Ser Leu His Ala Asn Met Lys Gly He 35 40 45 Val His Leu Asn Phe Leu Gin Val Pro Met Cys Lys Glu Tyr Glu Val 50 55 60 Lys Val He Gly Tyr Asn Leu Met Gin Ala Met Arg Phe Ala Val Glu 65 70 75 80 Glu He Asn Asn Asp Ser Ser Leu Leu Pro Gly Val Leu Leu Gly Tyr 85 90 95 Glu lie Val Asp Val Cys Tyr He Ser Asn Asn Val Gin Pro Val Leu 100 105 110 Tyr Phe Leu Ala His Glu Asp Asn Leu Leu Pro He Gin Glu Asp Tyr 115 120 125 Ser Asn Tyr He Ser Arg Val Val Ala Val He Gly Pro Asp Asn Ser 130 135 140 Glu Ser Val Met Thr Val Ala Asn Phe Leu Ser Leu Phe Leu Leu Pro 145 150 155 160 Gin He Thr Tyr Ser Ala He Ser Asp Glu Leu Arg Asp Lys Val Arg 165 170 175 Phe Pro Ala Leu Leu Arg Thr Thr Pro Ser Ala Asp His His Val Glu 180 185 190 Ala Met Val Gin Leu Met Leu His Phe Arg Trp Asn Trp He He Val 195 200 205 Leu Val Ser Ser Asp Thr Tyr Gly Arg Asp Asn Gly Gin Leu Leu Gly 210 215 220 Glu Arg Val Ala Arg Arg Asp lie Cys He Ala Phe Gin Glu Thr Leu 225 230 235 240 Pro Thr Leu Gin Pro Asn Gin Asn Met Thr Ser Glu Glu Arg Gin Arg 245 250 255 157 173,552/2

Leu Val Thr lie Val Asp Lys Leu Gin Gin Ser Thr Ala Arg Val Val 260 265 270 Val Val Phe Ser Pro Asp Leu Thr Leu Tyr His Phe Phe Asn Glu Val 275 280 285 Leu Arg Gin Asn Phe Thr Gly Ala Val Trp He Ala Ser Glu Ser Trp 290 295 300 Ala Xie Asp Pro Val Leu His Asn Leu Thr Glu Leu Gly His Leu Gly 305 310 315 320 Thr Phe Leu Gly He Thr He Gin Ser Val Pro He Pro Gly Phe Ser 325 330 335 Glu Phe Arg Glu Trp Gly Pro Gin Ala Gly Pro Pro Pro Leu Ser Arg 340 345 350 Thr Ser Gin Ser Tyr Thr Cys Asn Gin Glu Cys Asp Asn Cys Leu Asn 355 360 365 Ala Thr Leu Ser Phe Asn Thr He Leu Arg Leu Ser Gly Glu Arg Val 370 375 380 Val Tyr Ser Val Tyr Ser Ala Val Tyr Ala Val Ala His Ala Leu His 385 390 395 400 Ser Leu Leu Gly Cys Asp Lys Ser Thr Cys Thr Lys Arg Val Val Tyr 405 410 415 Pro Trp Gin Leu Leu Glu Glu He Trp Lys Val Asn Phe Thr Leu Leu 420 425 430 Asp His Gin He Phe Phe Asp Pro Gin Gly Asp Val Ala Leu His Leu 435 440 445 Glu lie Val Gin Trp Gin Trp Asp Arg Ser Gin Asn Pro Phe Gin Ser 450 455 460 Val Ala Ser Tyr Tyr Pro Leu Gin Arg Gin Leu Lys Asn lie Gin Asp 465 470 475 480 lie Ser Trp His Thr Val Asn Asn Thr He Pro Met Ser Met Cys Ser 485 490 495 Lys Arg Cys Gin Ser Gly Gin Lys Lys Lys Pro Val Gly He His Val 500 505 510 Cys Cys Phe Glu Cys He Asp Cys Leu Pro Gly Thr Phe Leu Asn His 515 520 525 Thr Glu Asp Glu Tyr Glu Cys Gin Ala Cys Pro Asn Asn Glu Trp Ser 530 535 540 Tyr Gin Ser Glu Thr Ser Cys Phe Lys Arg Gin Leu Val Phe Leu Glu 545 550 555 560 Trp His Glu Ala Pro Thr He Ala Val Ala Leu Leu Ala Ala Leu Gly 565 570 575 Phe Leu Ser Thr Leu Ala He Leu Val He Phe Trp Arg His Phe Gin 580 585 590 Thr Pro lie Val Arg Ser Ala Gly Gly Pro Met Cys Phe Leu Met Leu 595 600 605 Thr Leu Leu Leu Val Ala Tyr Met Val Val Pro Val Tyr Val Gly Pro 610 615 620 Pro Lys Val Ser Thr Cys Leu Cys Arg Gin Ala Leu Phe Pro Leu Cys 625 630 635 640 Phe Thr He Cys He Ser Cys lie Ala Val Arg Ser Phe Gin He Val 645 650 655 Cys Ala Phe Lys Met Ala Ser Arg Phe Pro Arg Ala Tyr Ser Tyr Trp 660 665 670 Val Arg Tyr Gin Gly Pro Tyr Val Ser Met Ala Phe He Thr Val Leu 675 680 685 Lys Met Val He Val Val He Gly Met Leu Ala Thr Gly Leu Ser Pro 690 695 700 Thr Thr Arg Thr Asp Pro Asp Asp Pro Lys He Thr He Val Ser Cys 705 710 715 720 Asn Pro Asn Tyr Arg Asn Ser Leu Leu Phe Asn Thr Ser Leu Asp Leu 725 730 735 158 173,552/2

Leu Leu Ser Val Val 740 Gly Phe Ser Phe 745 Ala Tyr Met Gly Lys 750 Glu Leu Pro Thr Asn Tyr Asn Glu Ala Lys Phe Ile Thr Leu Ser Met Thr Phe 755 760 765 Tyr Phe Thr Ser Ser Val Ser Leu Cys Thr Phe Met Ser Ala Tyr Ser 770 775 780 Gly Val Leu Val Thr Ile Val Asp Leu Leu Val Thr Val Leu Asn Leu 785 790 795 800 Leu Ala Ile Ser Leu Gly Tyr Phe Gly Pro Lys Cys Tyr Met Ile Leu 805 810 815 Phe Tyr Pro Glu Arg Asn Thr Pro Ala Tyr Phe Asn Ser Met Ile Gin 820 825 830 Gly Tyr Thr Met Arg Arg Asp 835 &lt;210&gt; 7 &lt;211&gt; 852

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 7 Met Leu Gly Pro Ala' Val Leu Gly Leu Ser Leu Trp Ala Leu Leu His 1 5 10 15 Pro Gly Thr Gly Ala Pro Leu Cys Leu Ser Gin Gin Leu Arg Met Lys 20 25 30 Gly Asp Tyr Val Leu Gly Gly Leu Phe Pro Leu Gly Glu Ala Glu Glu 35 40 45 Ala Gly Leu Arg Ser Arg Thr Arg Pro Ser Ser Pro Val Cys Thr Arg 50 55 60 Phe Ser Ser Asn Gly Leu Leu Trp Ala Leu Ala Met Lys Met Ala Val 65 70 75 80 Glu Glu Ile Asn Asn Lys Ser Asp Leu Leu Pro Gly Leu Arg Leu Gly 85 90 95 Tyr Asp Leu Phe Asp Thr Cys Ser Glu Pro Val Val Ala Met Lys Pro 100 105 110 Ser Leu Met Phe Leu Ala Lys Ala Gly Ser Arg Asp Ile Ala Ala Tyr 115 120 125 Cys Asn Tyr Thr Gin Tyr Gin Pro Arg Val Leu Ala Val Ile Gly Pro 130 135 140 His Ser Ser Glu Leu Ala Met Val Thr Gly Lys Phe Phe Ser Phe Phe 145 150 155 160 Leu Met Pro Gin Val· Ser Tyr Gly Ala Ser Met Glu Leu Leu Ser Ala 165 170 175 Arg Glu Thr Phe Pro Ser Phe Phe Arg Thr Val Pro Ser Asp Arg Val 180 185 190 Gin Leu Thr Ala Ala Ala Glu Leu Leu Gin Glu Phe Gly Trp Asn Trp 195 200 205 Val Ala Ala Leu Gly Ser Asp Asp Glu Tyr Gly Arg Gin Gly Leu Ser 210 215 220 He Phe Ser Ala Leu Ala Ala Ala Arg Gly Ile Cys Ile Ala His Glu 225 230 235 240 Gly Leu Val Pro Leu Pro Arg Ala Asp Asp Ser Arg Leu Gly Lys Val 245 250 255 Gin Asp Val Leu His Gin Val Asn Gin Ser Ser Val Gin Val Val Leu 260 265 270 Leu Phe Ala Ser Val His Ala Ala His Ala Leu Phe Asn Tyr Ser Ile 275 280 285 159 173,552/2

Ser Ser 290 Arg Leu Ser Pro Lys 295 Val Trp Val Ala Ser 300 Glu Ala Trp Leu Thr Ser Asp Leu Val Met Gly Leu Pro Gly Met Ala Gin Met Gly Thr 305 310 315 320 Val Leu Gly Phe Leu Gin Arg Gly Ala Gin Leu His Glu Phe Pro Gin 325 330 335 Tyr Val Lys Thr His Leu Ala Leu Ala Thr Asp Pro Ala Phe Cys Ser 340 345 350 Ala Leu Gly Glu Arg Glu Gin Gly Leu Glu Glu Asp Val Val Gly Gin 355 360 365 Arg Cys Pro Gin Cys Asp Cys Xie Thr Leu Gin Asn Val Ser Ala Gly 370 375 380 Leu Asn His His Gin Thr Phe Ser Val Tyr Ala Ala Val Tyr Ser Val 385 390 395 400 Ala Gin Ala Leu His Asn Thr Leu Gin Cys Asn Ala Ser Gly Cys Pro 405 410 415 Ala Gin Asp Pro Val Lys Pro Trp Gin Leu Leu Glu Asn Met Tyr Asn 420 425 430 Leu Thr Phe His Val Gly Gly Leu Pro Leu Arg Phe Asp Ser Ser Gly 435 440 445 Asn Val Asp Met Glu Tyr Asp Leu Lys Leu Trp Val Trp Gin Gly Ser 450 455 460 Val Pro Arg Leu His Asp Val Gly Arg Phe Asn Gly Ser Leu Arg Thr 465 470 475 480 Glu Arg Leu Lys Xie Arg Trp His Thr Ser Asp Asn Gin Lys Pro Val 485 490 495 Ser Arg Cys Ser Arg Gin Cys Gin Glu Gly Gin Val Arg Arg Val Lys 500 505 510 Gly Phe His Ser Cys Cys Tyr Asp Cys Val Asp Cys Glu Ala Gly Ser 515 520 525 Tyr Arg Gin Asn Pro Asp Asp lie Ala Cys Thr Phe Cys Gly Gin Asp 530 535 540 Glu Trp Ser Pro Glu Arg Ser Thr Arg Cys Phe Arg Arg Arg Ser Arg 545 550 555 560 Phe Leu Ala Trp Gly Glu Pro Ala Val Leu Leu Leu Leu Leu Leu Leu 565 570 575 Ser Leu Ala Leu Gly Leu Val Leu Ala Ala Leu Gly Leu Phe Val His 580 585 590 His Arg Asp Ser Pro Leu Val Gin Ala Ser Gly Gly Pro Leu Ala Cys 595 600 605 Phe Gly Leu Val Cys Leu Gly Leu Val Cys Leu Ser Val Leu Leu Phe 610 615 620 Pro Gly Gin Pro Ser Pro Ala Arg Cys Leu Ala Gin Gin Pro Leu Ser 625 630 635 640 His Leu Pro Leu Thr Gly Cys Leu Ser Thr Leu Phe Leu Gin Ala Ala 645 650 655 Glu He Phe Val Glu Ser Glu Leu Pro Leu Ser Trp Ala Asp Arg Leu 660 665 670 Ser Gly Cys Leu Arg Gly Pro Trp Ala Trp Leu Val Val Leu Leu Ala 675 680 685 Met Leu Val Glu Val Ala Leu Cys Thr Trp Tyr Leu Val Ala Phe Pro 690 695 700 Pro Glu Val Val Thr Asp Trp His Met Leu Pro Thr Glu Ala Leu Val 705 710 715 720 His Cys Arg Thr Arg Ser Trp Val Ser Phe Gly Leu Ala His Ala Thr 725 730 735 Asn Ala Thr Leu Ala Phe Leu Cys Phe Leu Gly Thr Phe Leu Val Arg 740 745 750 Ser Gin Pro Gly Arg Tyr Asn Arg Ala Arg Gly Leu Thr Phe Ala Met 755 760 765 160 173,552/1

Leu Ala Tyr Phe Ile Thr Trp Val Ser Phe Val Pro Leu Leu Ala Asn 770 775 780

Val Gin Val Val Leu Arg Pro Ala Val Gin Met Gly Ala Leu Leu Leu 785 790 795 800

Cys Val Leu Gly lie Leu Ala Ala Phe His Leu Pro Arg Cys Tyr Leu 805 810 815

Leu Met Arg Gin Pro Gly Leu Asn Thr Pro Glu Phe Phe Leu Gly Gly 820 825 830

Gly Pro Gly Asp Ala Gin Gly Gin Asn Asp Gly Asn Thr Gly Asn Gin 835 840 845

Gly Lys His Glu 850 &lt;210&gt; 8 &lt;211&gt; 2526

&lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 8 atgctgctct gcacggctcg cctggtcggc ctgcagcttc tcatttcctg ctgctgggcc 60 tttgcctgcc atagcacgga gtcttctcct gacttcaccc tccccggaga ttacctcctg 120 gcaggcctgt tccctctcca ttctggctgt ctgcaggtga ggcacagacc cgaggtgacc 180 ctgtgtgaca ggtcttgtag cttcaatgag catggctacc acctcttcca ggctatgcgg 240 cttggggttg aggagataaa caactccacg gccctgctgc ccaacatcac cctggggtac 300 cagctgtatg atgtgtgttc tgactctgcc aatgtgtatg ccacgctgag agtgctctcc 360 ctgccagggc aacaccacat agagctccaa ggagaccttc tccactattc ccctacggtg 420 ctggcagtga ttgggcctga cagcaccaac cgtgctgcca ccacagccgc cctgctgagc 480 cctttcctgg tgcccatgat tagctatgcg gccagcagcg agacgctcag cgtgaagcgg 540 cagtatccct ctttcctgcg caccatcccc aatgacaagt accaggtgga gaccatggtg 600 ctgctgctgc agaagttcgg gtggacctgg atctctctgg ttggcagcag tgacgactat 660 gggcagctag gggtgcaggc actggagaac caggccactg gtcaggggat ctgcattgct 720 ttcaaggaca tcatgccctt ctctgcccag gtgggcgatg agaggatgca gtgcctcatg 780 cgccacctgg cccaggccgg ggccaccgtc gtggttgttt tttccagccg gcagttggcc 840 agggtgtttt tcgagtccgt ggtgctgacc aacctgactg gcaaggtgtg ggtcgcctca 900 gaagcctggg ccctctccag gcacatcact ggggtgcccg ggatccagcg cattgggatg 960 gtgctgggcg tggccatcca gaagagggct gtccctggcc tgaaggcgtt tgaagaagcc 1020 tatgcccggg cagacaagaa ggcccctagg ccttgccaca agggctcctg gtgcagcagc 1080 aatcagctct gcagagaatg ccaagctttc atggcacaca cgatgcccaa gctcaaagcc 1140 ttctccatga gttctgccta caacgcatac cgggctgtgt atgcggtggc ccatggcctc 1200 caccagctcc tgggctgtgc ctctggagct tgttccaggg gccgagtcta cccctggcag 1260 cttttggagc agatccacaa ggtgcatttc cttctacaca aggacactgt ggcgtttaat 1320 gacaacagag atcccctcag tagctataac ataattgcct gggactggaa tggacccaag 1380 tggaccttca cggtcctcgg ttcctccaca tggtctccag ttcagctaaa cataaatgag 1440 accaaaatcc agtggcacgg aaaggacaac caggtgccta agtctgtgtg ttccagcgac 1500 tgtcttgaag ggcaccagcg agtggttacg ggtttccatc actgctgctt tgagtgtgtg 1560 ccctgtgggg ctgggacctt cctcaacaag agtgacctct acagatgcca gccttgtggg 1620 aaagaagagt gggcacctga gggaagccag acctgcttcc cgcgcactgt ggtgtttttg 1680 gctttgcgtg agcacacctc ttgggtgctg ctggcagcta acacgctgct gctgctgctg 1740 ctgcttggga ctgctggcct gtttgcctgg cacctagaca cccctgtggt gaggtcagca 1800 gggggccgcc tgtgctttct tatgctgggc tccctggcag caggtagtgg cagcctctat 1860 ggcttctttg gggaacccac aaggcctgcg tgcttgctac gccaggccct ctttgccctt 1920 ggtttcacca tcttcctgtc ctgcctgaca gttcgctcat tccaactaat catcatcttc 1980 aagttttcca ccaaggtacc tacattctac cacgcctggg tccaaaacca cggtgctggc 2040 ctgtttgtga tgatcagctc agcggcccag ctgcttatct gtctaacttg gctggtggtg 2100 tggaccccac tgcctgctag ggaataccag cgcttccccc atctggtgat gcttgagtgc 2160 acagagacca actccctggg cttcatactg gccttcctct acaatggcct cctctccatc 2220 agtgcctttg cctgcagcta cctgggtaag gacttgccag agaactacaa cgaggccaaa 2280 161 173,552/1 tgtgtcacct tcagcctgct cttcaacttc gtgtcctgga tcgccttctt caccacggcc 2340 agcgtctacg acggcaagta cctgcctgcg gccaacatga tggctgggct gagcagcctg 2400 agcagcggct tcggtgggta ttttctgcct aagtgctacg tgatcctctg ccgcccagac 2460 ctcaacagca cagagcactt ccaggcctcc attcaggact acacgaggcg ctgcggctcc 2520 acctga 2526 &lt;210&gt; 9 &lt;211&gt; 2559

&lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 9 atgctgggcc ctgctgtcct gggcctcagc ctctgggctc tcctgcaccc tgggacgggg 60 gccccattgt gcctgtcaca gcaacttagg atgaaggggg actacgtgct gggggggctg 120 ttccccctgg gcgaggccga ggaggctggc ctccgcagcc ggacacggcc cagcagccct 180 gtgtgcacca ggttctcctc aaacggcctg ctctgggcac tggccatgaa aatggccgtg 240 gaggagatca acaacaagtc ggatctgctg cccgggctgc gcctgggcta cgacctcttt 300 gatacgtgct cggagcctgt ggtggccatg aagcccagcc tcatgttcct, ggccaaggca 360 ggcagccgcg acatcgccgc ctactgcaac tacacgcagt accagccccg tgtgctggct 420 gtcatcgggc cccactcgtc agagctcgcc atggtcaccg gcaagttctt cagcttcttc 480 ctcatgcccc aggtcagcta cggtgctagc atggagctgc tgagcgcccg ggagaccttc 540 ccctccttct tccgcaccgt gcccagcgac cgtgtgcagc tgacggccgc cgcggagctg 600 ctgcaggagt tcggctggaa ctgggtggcc gccctgggca gcgacgacga gtacggccgg 660 cagggcctga gcatcttctc ggccctggcc gcggcacgcg gcatctgcat cgcgcacgag 720 ggcctggtgc cgctgccccg tgccgatgac tcgcggctgg ggaaggtgca ggacgtcctg 780 caccaggtga accagagcag cgtgcaggtg gtgctgctgt tcgcctccgt gcacgccgcc 840 cacgccctct tcaactacag catcagcagc aggctctcgc ccaaggtgtg ggtggccagc 900 gaggcctggc tgacctctga cctggtcatg gggctgcccg gcatggccca gatgggcacg 960 gtgcttggct tcctccagag gggtgcccag ctgcacgagt tcccccagta cgtgaagacg 1020 cacctggccc tggccaccga cccggccttc tgctctgccc tgggcgagag ggagcagggt 1080 ctggaggagg acgtggtggg ccagcgctgc ccgcagtgtg actgcatcac gctgcagaac 1140 gtgagcgcag ggctaaatca ccaccagacg ttctctgtct acgcagctgt gtatagcgtg 1200 gcccaggccc tgcacaacac tcttcagtgc aacgcctcag gctgccccgc gcaggacccc 1260 gtgaagccct ggcagctcct ggagaacatg tacaacctga ccttccacgt gggcgggctg 1320 ccgctgcggt tcgacagcag cggaaacgtg gacatggagt acgacctgaa gctgtgggtg 1380 tggcagggct cagtgcccag gctccacgac gtgggcaggt tcaacggcag cctcaggaca 1440 gagcgcctga agatccgctg gcacacgtct gacaaccaga agcccgtgtc ccggtgctcg 1500 cggcagtgcc aggagggcca ggtgcgccgg gtcaaggggt tccactcctg ctgctacgac 1560 tgtgtggact gcgaggcggg cagctaccgg caaaacccag acgacatcgc ctgcaccttt 1620 tgtggccagg atgagtggtc cccggagcga agcacacgct gcttccgccg caggtctcgg 1680 ttcctggcat ggggcgagcc ggctgtgctg ctgctgctcc tgctgctgag cctggcgctg 1740 ggccttgtgc tggctgcttt ggggctgttc gttcaccatc gggacagccc actggttcag 1800 gcctcggggg ggcccctggc ctgctttggc ctggtgtgcc tgggcctggt ctgcctcagc 1860 gtcctcctgt tccctggcca gcccagccct gcccgatgcc tggcccagca gcccttgtcc 1920 cacctcccgc tcacgggctg cctgagcaca ctcttcctgc aggcggccga gatcttcgtg 1980 gagtcagaac tgcctctgag ctgggcagac cggctgagtg gctgcctgcg ggggccctgg 2040 gcctggctgg tggtgctgct ggccatgctg gtggaggtcg cactgtgcac ctggtacctg 2100 gtggccttcc cgccggaggt ggtgacggac tggcacatgc tgcccacgga ggcgctggtg 2160 cactgccgca cacgctcctg ggtcagcttc ggcctagcgc acgccaccaa tgccacgctg 2220 gcctttctct gcttcctggg cactttcctg gtgcggagcc agccgggctg ctacaaccgt 2280 gcccgtggcc tcacctttgc catgctggcc tacttcatca cctgggtctc ctttgtgccc 2340 ctcctggcca atgtgcaggt ggtcctcagg cccgccgtgc agatgggcgc cctcctgctc 2400 tgtgtcctgg gcatcctggc tgccttccac ctgcccaggt gttacctgct catgcggcag 2460 ccagggctca acacccccga gttcttcctg ggagggggcc ctggggatgc ccaaggccag 2520 aatgacggga acacaggaaa tcaggggaaa catgagtga 2559 162 173,552/1 &lt;210&gt; 10 &lt;211&gt; 2518

&lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 10 atggggccca gggcaaagac catctgctcc ctgttcttcc tcctatgggt cctggctgag 60 ccggctgaga actcggactt ctacctgcct ggggattacc tcctgggtgg cctcttctcc 120 ctccatgcca acatgaaggg cattgttcac cttaacttcc tgcaggtgcc catgtgcaag 180 gagtatgaag tgaaggtgat aggctacaac ctcatgcagg ccatgcgctt cgcggtggag 240 gagatcaaca atgacagcag cctgctgcct ggtgtgctgc tgggctatga gatcgtggat 300 gtgtgctaca tctccaacaa tgtccagccg gtgctctact tcctggcaca cgaggacaac 360 ctccttccca tccaagagga ctacagtaac tacatttccc gtgtggtggc tgtcattggc 420 cctgacaact ccgagtctgt catgactgtg gccaattcct ctccctattt ctccttccac 480 agatcaccta cagcgccatc agcgatgagc tgcgagacaa ggtgcgcttc ccggctttgc 540 tgcgtaccac acccagcgcc gaccaccacg tcgaggccat ggtgcagctg atgctgcact 600 tccgctggaa ctggatcatt gtgctggtga gcagcgacac ctatggccgc gacaatggca 660 gctgcttggc gagcgcgtgg cccggcgcga catctgcatc gccttccagg agacgctgcc 720 cacactgcag cccaaccaga acatgacgtc agaggagcgc cagcgcctgg tgaccattgt 780 ggacaagctg cagcagagca cagcgcgcgt cgtggtcgtg ttctcgcccg acctgaccct 840 gtaccacttc ttcaatgagg tgctgcgcca gaacttcacg ggcgccgtgt ggatcgcctc 900 cgagtcctgg gccatcgacc cggtcctgca caacctcacg gagctgggcc acttgggcac 960 cttcctgggc atcaccatcc agagcgtgcc catcccgggc ttcagtgagt tccgcgagtg 1020 gggcccacag gctgggccgc cacccctcag chggaccagc cagagctata cctgcaacca 1080 ggagtgcgac aactgcctga acgccacctt gtccttcaac accattctca ggctctctgg 1140 ggagcgtgtc gtctacagcg tgtactctgc ggtctatgct gtggcccatg ccctgcacag 1200 cctcctcggc tgtgacaaaa gcacctgcac caagagggtg gtctacccct ggcagctgct 1260 tgaggagatc tggaaggtca acttcactct cctggaccac caaatcttct tcgacccgca 1320 aggggacgtg gctctgcact tggagattgt ccagtggcaa tgggaccgga gccagaatcc 1380 cttccagagc gtcgcctcct actaccccct gcagcgacag ctgaagaaca tccaagacat 1440 ctcctggcac accgtcaaca acacgatccc tatgtccatg tgttccaaga ggtgccagtc 1500 agggcaaaag aagaagcctg tgggcatcca cgtctgctgc ttcgagtgca tcgactgcct 1560 tcccggcacc ttcctcaacc acactgaaga tgaatatgaa tgccaggcct gcccgaataa 1620 cgagtggtcc taccagagtg agacctcctg cttcaagcgg cagctggtct tcctggaatg 1680 gcatgaggca cccaccatcg ctgtggccct gctggccgcc ctgggcttcc tcagcaccct 1740 ggccatcctg gtgatattct ggaggcactt ccagacaccc atagttcgct cggctggggg 1800 ccccatgtgc ttcctgatgc tgacactgct gctggtggca tacatggtgg tcccggtgta 1860 cgtggggccg cccaaggtct ccacctgcct ctgccgccag gccctctttc ccctctgctt 1920 cacaatttgc atctcctgta tcgccgtgcg ttctttccag atcgtctgcg ccttcaagat 1980 ggccagccgc ttcccacgcg cctacagcta ctgggtccgc taccaggggc cctacgtctc 2040 tatggcattt atcacggtac tcaaaatggt cattgtggta attggcatgc tggccacggg 2100 cctcagtccc accacccgta ctgaccccga tgaccccaag atcacaattg tctcctgtaa 2160 ccccaactac cgcaacagcc tgctgttcaa caccagcctg gacctgctgc tctcagtggt 2220 gggtttcagc ttcgcctaca tgggcaaaga gctgcccacc aactacaacg aggccaagtt 2280 catcaccctc agcatgacct tctatttcac ctcatccgtc tccctctgca ccttcatgtc 2340 tgcctacagc ggggtgctgg tcaccatcgt ggacctcttg gtcactgtgc tcaacctcct 2400 ggccatcagc ctgggctact tcggccccaa gtgctacatg atcctcttct acccggagcg 2460 caacacgccc gcctacttca acagcatgat ccagggctac accatgagga gggactag 2518 &lt;210&gt; 11 &lt;211&gt; 2577

&lt;212&gt; DNA &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note'= synthetic construct 163 173,552/1 &lt;400&gt; 11 atgccgggtt tggctatctt gggcctcagt ctggctgctt tcctggagct tgggatgggg 60 tcctctttgt gtctgtcaca gcaattcaag gcacaagggg actatatatt gggtggacta 120 tttcccctgg gcacaactga ggaggccact ctcaaccaga gaacacagcc caacggcatc 180 ctatgtacca ggttctcgcc ccttggtttg ttcctggcca tggctatgaa gatggctgta 240 gaggagatca acaatggatc tgccttgctc cctgggctgc gactgggcta tgacctgttt 300 gacacatgct cagagccagt ggtcaccatg aagcccagcc tcatgttcat ggccaaggtg 360 ggaagtcaaa gcattgctgc ctactgcaac tacacacagt accaaccccg tgtgctggct 420 gtcattggtc cccactcatc agagcttgcc ctcattacag gcaagttctt cagcttcttc 480 ctcatgccac aggtcagcta tagtgccagc atggatcggc taagtgaccg ggaaacattt 540 ccatccttct tccgcacagt gcccagtgac cgggtgcagc tgcaggccgt tgtgacactg 600 ttgcagaatt tcagctggaa ctgggtggct gccttaggta gtgatgatga ctatggccgg 660 gaaggtctga gcatcttttc tggtctggcc aactcacgag gtatctgcat tgcacacgag 720 ggcctggtgc cacaacatga cactagtggc caacaattgg gcaaggtggt ggatgtgcta 780 cgccaagtga accaaagcaa agtacaggtg gtggtgctgt ttgcatctgc ccgtgctgtc 840 tactcccttt ttagctacag catccttcat gacctctcac ccaaggtatg ggtggccagt 900 gagtcctggc tgacctctga cctggtcatg acacttccca atattgcccg tgtgggcact 960 gttcttgggt ttctgcagcg cggtgcccta ctgcctgaat tttcccatta tgtggagact 1020 cgccttgccc tagctgctga cccaacattc tgtgcctccc tgaaagctga gttggatctg 1080 gaggagcgcg tgatggggcc acgctgttca caatgtgact acatcatgct acagaacctg 1140 tcatctgggc tgatgcagaa cctatcagct gggcagttgc accaccaaat atttgcaacc 1200 tatgcagctg tgtacagtgt ggctcaggcc cttcacaaca ccctgcagtg caatgtctca 1260 cattgccaca catcagagcc tgttcaaccc tggcagctcc tggagaacat gtacaatatg 1320 agtttccgtg ctcgagactt gacactgcag tttgatgcca aagggagtgt agacatggaa 1380 tatgacctga agatgtgggt gtggcagagc cctacacctg tactacatac tgtaggcacc 1440 ttcaacggca cccttcagct gcagcactcg aaaatgtatt ggccaggcaa ccaggtgcca 1500 gtctcccagt gctcccggca gtgcaaagat ggccaggtgc gcagagtaaa gggctttcat 1560 tcctgctgct atgactgtgt ggactgcaag gcagggagct accggaagca tccagatgac 1620 ttcacctgta ctccatgtgg caaggatcag tggtccccag aaaaaagcac aacctgctta 1680 cctcgcaggc ccaagtttct ggcttggggg gagccagctg tgctgtcact tctcctgctg 1740 ctttgcctgg tgctgggcct gacactggct gccctggggc tctttgtcca ctactgggac 1800 agccctcttg ttcaggcctc aggtgggtca ctgttctgct ttggcctgat ctgcctaggc 1860 ctcttctgcc tcagtgtcct tctgttccca ggacgaccac gctctgccag ctgccttgcc 1920 caacaaccaa tggctcacct ccctctcaca ggctgcctga gcacactctt cctgcaagca 1980 gccgagatct ttgtggagtc tgagctgcca ctgagttggg caaactggct ctgcagctac 2040 cttcggggcc cctgggcttg gctggtggta ctgctggcca ctcttgtgga ggctgcacta 2100 tgtgcctggt acttgatggc tttccctcca gaggtggtga cagattggca ggtgctgccc 2160 acggaggtac tggaacactg ccgcatgcgt tcctgggtca gcctgggctt ggtgcacatc 2220 accaatgcag tgttagcttt cctctgcttt ctgggcactt tcctggtaca gagccagcct 2280 ggtcgctata accgtgcccg tggcctcacc ttcgccatgc tagcttattt catcatctgg 2340 gtctcttttg tgcccctcct ggctaatgtg caggtggcct accagccagc tgtgcagatg 2400 ggtgctatct tattctgtgc cctgggcatc ctggccacct tccacctgcc caaatgctat 2460 gtacttctgt ggctgccaga gctcaacacc caggagttct tcctgggaag gagccccaag 2520 gaagcatcag atgggaatag tggtagtagt gaggcaactc ggggacacag tgaatga 2577 &lt;210&gt; 12 &lt;211&gt; 137

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 12

Pro Ser Pro Phe Arg Asp Ile Val Ser Tyr Pro Asp Lys Ile Ile Leu 15 10 15

Gly Cys Phe Met Asn Leu Lys Thr Ser Ser Val Ser Phe Val Leu Leu 20 25 30 164 173,552/1

Leu Leu Leu 35 Cys Leu Leu Cys Phe 40 He Phe Ser Tyr Met 45 Gly Lys Asp Leu Pro Lys Asn Tyr Asn Glu Ala Lys Ala He Thr Phe Cys Leu Leu 50 55 60 Leu Leu He Leu Thr Trp He He Phe Thr Thr Ala Ser Leu Leu Tyr 65 70 75 80 Gin Gly Lys Tyr He His Ser Leu Asn Ala Leu Ala Val Leu Ser Ser 85 90 95 lie Tyr Ser Phe Leu Leu Trp Tyr Phe Leu Pro Lys Cys Tyr He lie 100 105 110 He Phe Gin Pro Gin Lys Asn Thr Gin Lys Tyr Phe Gin Gly Leu He 115 120 125 Gin Asp Tyr Thr Lys Thr He Ser Gin 130 135 &lt;210&gt; 13 &lt;211&gt; 242

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial synthetic construct Sequence; note = &lt;221&gt; VARIANT &lt;222&gt; 120,121 &lt;223&gt; Xaa = any amino acid &lt;400&gt; 13 Phe Ala Val Asn Tyr Asn Thr Pro Val Val Arg Ser Ala Gly Gly Pro 1 5 10 15 Met Cys Phe Leu lie Leu Gly Cys Leu Ser Leu Cys Ser He Ser Val 20 25'· 30 Phe Phe Tyr Phe Glu Arg Pro Thr Glu Ala Phe Cys He Leu Arg Phe 35 40 45 Met Pro Phe Leu Leu Phe Tyr Ala Val Cys Leu Ala Cys Phe Ala Val 50 55 60 Arg Ser Phe Gin He Val He He Phe Lys He Ala Ala Lys Phe Pro 65 70 75 80 Arg Val His Ser Trp Trp Met Lys Tyr His Gly Gin Trp Leu Val He 85 90 95 Ser Met Thr Phe Val Leu Gin Ala Val Val He Val He Gly Phe Ser 100 105 110 Ser Asn Pro Pro Leu Pro Tyr Xaa Xaa Phe Val Ser Tyr Pro Asp Lys 115 120 125 He He Leu Gly Cys Asp Val Asn Leu Asn Met Ala Ser Thr Ser Phe 130 135 140 Phe Leu Leu Leu Leu Leu Cys lie Leu Cys Phe Thr Phe Ser Tyr Met 145 150 155 160 Gly Lys Asp Leu Pro Lys Asn Tyr Asn Glu Ala Lys Ala He Thr Phe 165 170 175 Cys Leu Leu Leu Leu He Leu Thr Trp He He Phe Ala Thr Ala Phe 180 185 190 Met Leu Tyr His Gly Lys Tyr He His Thr Leu Asn Ala Leu Ala Val 195 200 205 Leu Ser Ser Ala Tyr Cys Phe Leu Leu Trp Tyr Phe Leu Pro Lys Cys 210 215 220 Tyr He He He Phe Gin Pro His Lys Asn Thr Gin Lys Tyr Phe Gin 225 230 235 240 Leu Ser 165 173,552/1 &lt;210&gt; 14 &lt;211&gt; 165

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial synthetic construct Sequence; note = &lt;400&gt; 14 Lys Lys Gin Gly Pro Glu Val Asp lie Phe He Val Ser Val Thr He 1 5 10 15 Leu Cys lie Ser Val Leu Gly Val Ala Val Gly Pro Pro Glu Pro Ser 20 25 30 Gin Asp Leu Asp Phe Tyr Met Asp Ser He Val Leu Glu Cys Ser Asn 35 40 45 Thr Leu Ser Pro Gly Ser Phe He Glu Leu Cys Tyr Val Cys Val Leu 50 55 60 Ser Val Leu Cys Phe Phe Phe Ser Tyr Met Gly Lys Asp Leu Pro Ala 65 70 75 80 Asn Tyr Asn Glu Ala Lys Cys Val Thr Phe Ser Leu Met Val Tyr Met 85 90 95 He Ser Trp He Ser Phe Phe Thr Val Tyr Leu He Ser Arg Gly Pro 100 105 110 Phe Thr Val Ala Ala Tyr Val Cys Ala Thr Leu Val Ser Val Leu Ala 115 120 125 Phe Phe Gly Gly Tyr Phe Leu Pro Lys He Tyr lie lie Val Leu Lys 130 135 140 Pro Gin Met Asn Thr Thr Ala His Phe Gin Asn Cys lie Gin Met Tyr 145 150 155 160 Thr Met Ser Lys Gin 165 &lt;210&gt; 15 &lt;211&gt; 236

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct

&lt;221&gt; VARIANT &lt;222&gt; 8,15,59,62,76,117, 128,136,168,173,175,176,203,226 &lt;223&gt; Xaa = any animo acid &lt;400&gt; 15

Ala Pro Lys Ser Ser Gin Arg Xaa Leu Arg Arg Thr Arg Leu Xaa Leu 1 5 10 15 Glu Trp Asp His Pro Met Ser Val Ala Leu Leu Phe Phe Leu Val Cys 20 25 30 Cys Leu Leu Met Thr Ser Ser Ser Ala Val He Leu Leu Leu Asn He 35 40 45 Asn Thr Pro Val Ala Lys Ser Ala Gly Gly Xaa Thr Cys Xaa Leu Lys 50 55 60 Leu Ala Ala Leu Thr Ala Ala Ala Met Ser Ser Xaa Cys His Phe Gly 65 70 75 80 Gin Pro Ser Pro Leu Ala Ser Lys Leu Lys Gin Pro Gin Phe Thr Phe 85 90 95 166 173,552/1

Ser Phe Thr Val 100 Cys Leu Ala Cys Asn 105 Arg Cys Ala Leu Ala 110 Thr Gly His Leu His Phe Xaa Ile Arg Val Ala Leu Pro Pro Ala Tyr Asn Xaa 115 120 125 Trp Ala Lys Asn His Gly Pro Xaa Ala Thr Ile Phe Ile Ala Ser Ala 130 135 140 Ala Ile Leu Cys Val Leu Cys Leu Arg Val Ala Val Gly Pro Pro Gin 145 150 155 160 Pro Ser Gin Asx Leu Asx Phe Xaa Thr Asn Ser lie Xaa Leu Xaa Xaa 165 170 175 Ser Asn Thr Leu Ser Pro Gly Ser Phe Val Glu Leu Cys Asn Val Ser 180 185 190 Leu Leu Ser Ala Val Cys Phe Val Phe Ser Xaa Met Gly Lys Asx Leu 195 200 205 Pro Ala Asn Tyr Asn Glu Ala Lys Cys Val Thr Phe Ser Leu Met Val 210 215 220 Asn Xaa lie Ser Trp Ile Ser Phe Phe Thr Val Tyr 225 230 235 &lt;210&gt; 16 &lt;211&gt; 838

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 16

Met Gly Pro Arg Ala Lys Thr Ile Cys Ser Leu Phe Phe Leu Leu Trp 1 5 10 15 Val Leu Ala Glu Pro Ala Glu Asn Ser Asp Phe Tyr Leu Pro Gly Asp 20 25 30 Tyr Leu Leu Gly Gly Leu Phe Ser Leu His Ala Asn Met Lys Gly Ile 35 40 45 Val His Leu Asn Phe Leu Gin Val Pro Met Cys Lys Glu Tyr Glu Val 50 55 60 Lys Val Ile Gly Tyr Asn Leu Met Gin Ala Met Arg Phe Ala Val Glu 65 70 75 80 Glu Ile Asn Asn Asp Ser Ser Leu Leu Pro Gly Val Leu Leu Gly Tyr 85 90 95 Glu Ile Val Asp Val Cys Tyr Ile Ser Asn Asn Val Gin Pro Val Leu 100 105 110 Tyr Phe Leu Ala His Glu Asp Asn Leu Leu Pro Ile Gin Glu Asp Tyr 115 120 125 Ser Asn Tyr Ile Ser Arg Val Val Ala Val Ile Gly Pro Asp Asn Ser 130 135 140 Glu Ser Val Met Thr Val Ala Asn Phe Leu Ser Leu Phe Leu Leu Pro 145 150 155 160 Gin Ile Thr Tyr Ser Ala lie Ser Asp Glu Leu Arg Asp Lys Val Arg 165 170 175 Phe Pro Ala Leu Leu Arg Thr Thr Pro Ser Ala Asp His His Val Glu 180 185 190 Ala Met Val Gin Leu Met Leu His Phe Arg Trp Asn Trp Ile Ile Val 195 200 205 Leu Val Ser Ser Asp Thr Tyr Gly Arg Asp Asn Gly Gin Leu Leu Gly 210 215 220 Glu Arg Val Ala Arg Arg Asp Ile Cys Ile Ala Phe Gin Glu Thr Leu 225 230 235 240 Pro Thr Leu Gin Pro Asn Gin Asn Met Thr Ser Glu Glu Arg Gin Arg 245 250 255 167 173,552/1

Leu Val Thr He 260 Val Asp Lys Leu Gin Gin 265 Ser Thr Ala Arg 270 Val Val Val Val Phe Ser Pro Asp Leu Thr Leu Tyr His Phe Phe Asn Glu Val 275 280 285 Leu Arg Gin Asn Phe Thr Gly Ala Val Trp He Ala Ser Glu Ser Trp 290 295 300 Ala He Asp Pro Val Leu His Asn Leu Thr Glu Leu Gly His Leu Gly 305 310 315 320 Thr Phe Leu Gly He Thr He Gin Ser Val Pro He Pro Gly Phe Ser 325 330 335 Glu Phe Arg Glu Trp Gly Pro Gin Ala Gly Pro Pro Pro Leu Ser Arg 340 345 350 Thr Ser Gin Ser Tyr Thr Cys Asn Gin Glu Cys Asp Asn Cys Leu Asn 355 360 365 Ala Thr Leu Ser Phe Asn Thr lie Leu Arg Leu Ser Gly Glu Arg Val 370 375 380 Val Tyr Ser Val Tyr Ser Ala Val Tyr Ala Val Ala His Ala Leu His 385 390 395 400 Ser Leu Leu Gly Cys Asp Lys Ser Thr Cys Thr Lys Arg Val Val Tyr 405 410 415 Pro Trp Gin Leu Leu Glu Glu He Trp Lys Val Asn Phe Thr Leu Leu 420 425 430 Asp His Gin He Phe Phe Asp Pro Gin Gly Asp Val Ala Leu His Leu 435 440 445 Glu He Val Gin Trp Gin Trp Asp Arg Ser Gin Asn Pro Phe Gin Ser 450 455 460 Val Ala Ser Tyr Tyr Pro Leu Gin Arg Gin Leu Lys Asn He Gin Asp 465 470 475 480 lie Ser Trp His Thr Val Asn Asn Thr He Pro Met Ser Met Cys Ser 485 490 495 Lys Arg Cys Gin Ser Gly Gin Lys Lys Lys Pro Val Gly He His Val 500 505 510 Cys Cys Phe Glu Cys He Asp Cys Leu Pro Gly Thr Phe Leu Asn His 515 520 525 Thr Glu Asp Glu Tyr Glu Cys Gin Ala Cys Pro Asn Asn Glu Trp Ser 530 535 540 Tyr Gin Ser Glu Thr Ser Cys Phe Lys Arg Gin Leu Val Phe Leu Glu 545 550 555 560 His Glu Val Pro Thr lie Val Val Ala He Leu Ala Ala Leu Gly Phe 565 570 575 Phe Ser Thr Leu Ala He Leu Phe He Phe Trp Arg His Phe Gin Thr 580 585 590 Pro Met Val Arg Ser Ala Gly Gly Pro Met Cys Phe Leu Met Leu Val 595 600 605 Pro Leu Leu Leu Ala Phe Gly Met Val Pro Val Tyr Val Gly Pro Pro 610 615 620 Thr Val Phe Ser Cys Phe Cys Arg Gin Ala Phe Phe Thr Val Cys Phe 625 630 635 640 Ser He Cys Leu Ser Cys He Thr Val Arg Ser Phe Gin He Val Cys 645 650 655 Val Phe Lys Met Ala Arg Arg Leu Pro Ser Ala Tyr Ser Phe Trp Met 660 665 670 Arg Tyr His Gly Pro Tyr Val Phe Val Ala Phe He Thr Ala lie Lys 675 680 685 Val Ala Leu Val Val Gly Asn Met Leu Ala Thr Thr He Asn Pro He 690 695 700 Gly Arg Thr Asp Pro Asp Asp Pro Asn He Met lie Leu Ser Cys His 705 710 715 720 Pro Asn Tyr Arg Asn Gly Leu Leu Phe Asn Thr Ser Met Asp Leu Leu 725 730 735 168 173,552/1

Leu Ser Val Leu 740 Gly Phe Ser Phe Ala Tyr 745 Met Gly Lys Glu 750 Leu Pro Thr Asn Tyr Asn Glu Ala Lys Phe Ile Thr Leu Ser Met Thr Phe Ser 755 760 765 Phe Thr Ser Ser lie Ser Leu Cys Thr Phe Met Ser Val His Asp Gly 770 775 780 Val Leu Val Thr Ile Met Asp Leu Leu Val Thr Val Leu Asn Phe Leu 785 790 795 800 Ala Ile Gly Leu Gly Tyr Phe Gly Pro Lys Cys Tyr Met Ile Leu Phe 805 810 815 Tyr Pro Glu Arg Asn Thr Ser Ala Tyr Phe Asn Ser Met Ile Gin Gly 820 825 830 Tyr Thr Met Arg Lys Ser 835 &lt;210&gt; 17 &lt;211&gt; 844

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 17

Met Gly Pro Gin Ala Arg Thr Leu Cys Leu Leu Ser Leu Leu Leu His 1 5 10 15 Val Leu Pro Lys Pro Gly Lys Leu Val Glu Asn Ser Asp Phe His Leu 20 25 30 Ala Gly Asp Tyr Leu Leu Gly Gly Leu Phe Thr Leu His Ala Asn Val 35 40 45 Lys Ser Ile Ser His Leu Ser Tyr Leu Gin Val Pro Lys Cys Asn Glu 50 55 60 Phe Thr Met Lys Val Leu Gly Tyr Asn Leu Met Gin Ala Met Arg Phe 65 70 75 80 Ala Val Glu Glu Ile Asn Asn Cys Ser Ser Leu Leu Pro Gly Val Leu 85 90 95 Leu Gly Tyr Glu Met Val Asp Val Cys Tyr Leu Ser Asn Asn Ile His 100 105 110 Pro Gly Leu Tyr Phe Leu Ala Gin Asp Asp Asp Leu Leu Pro Ile Leu 115 120 125 Lys Asp Tyr Ser Gin Tyr Met Pro His Val Val Ala Val Ile Gly Pro 130 135 140 Asp Asn Ser Glu Ser Ala lie Thr Val Ser Asn Ile Leu Ser His Phe 145 150 155 160 Leu Ile Pro Gin Ile Thr Tyr Ser Ala Ile Ser Asp Lys Leu Arg Asp 165 170 175 Lys Arg His Phe Pro Ser Met Leu Arg Thr Val Pro Ser Ala Thr His 180 185 190 His Ile Glu Ala Met Val Gin Leu Met Val His Phe Gin Trp Asn Trp 195 200 205 Ile Val Val Leu Val Ser Asp Asp Asp Tyr Gly Arg Glu Asn Ser His 210 215 220 Leu Leu Ser Gin Arg Leu Thr Lys Thr Ser Asp Ile Cys Ile Ala Phe 225 230 235 240 Gin Glu Val Leu Pro Ile Pro Glu Ser Ser Gin Val Met Arg Ser Glu 245 250 2 55 Glu Gin Arg Gin Leu Asp Asn Ile Leu Asp Lys Leu Arg Arg Thr Ser 260 265 270 Ala Arg Val Val Val Val Phe Ser Pro Glu Leu Ser Leu Tyr Ser Phe 275 280 285 169 173,552/1

Phe His 290 Glu Val Leu Arg Trp Asn 295 Phe Thr Gly Phe 300 Val Trp lie Ala Ser Glu Ser Trp Ala He Asp Pro Val Leu His Asn Leu Thr Glu Leu 305 310 315 320 Arg His Thr Gly Thr Phe Leu Gly Val Thr He Gin Arg Val Ser lie 325 330 335 Pro Gly Phe Ser Gin Phe Arg Val Arg Arg Asp Lys Pro Gly Tyr Pro 340 345 350 Val Pro Asn Thr Thr Asn Leu Arg Thr Thr Cys Asn Gin Asp Cys Asp 355 360 365 Ala Cys Leu Asn Thr Thr Lys Ser Phe Asn Asn He Leu He Leu Ser 370 375 380 Gly Glu Arg Val Val Tyr Ser Val Tyr Ser Ala Val Tyr Ala Val Ala 385 390 395 400 His Ala Leu His Arg Leu Leu Gly Cys Asn Arg Val Arg Cys Thr Lys 405 410 415 Gin Lys Val Tyr Pro Trp Gin Leu Leu Arg Glu lie Trp His Val Asn 420 425 430 Phe Thr Leu Leu Gly Asn Arg Leu Phe Phe Asp Gin Gin Gly Asp Met 435 440 445 Pro Met Leu Leu Asp He lie Gin Trp Gin Trp Asp Leu Ser Gin Asn 450 455 460 Pro Phe Gin Ser He Ala Ser Tyr Ser Pro Thr Ser Lys Arg Leu Thr 465 470 475 480 Tyr lie Asn Asn Val Ser Trp Tyr Thr Pro Asn Asn Thr Val Pro Val 485 490 495 Ser Met Cys Ser Lys Ser Cys Gin Pro Gly Gin Met Lys Lys Ser Val 500 505 510 Gly Leu His Pro Cys Cys Phe Glu Cys Leu Asp Cys Met Pro Gly Thr 515 520 525 Tyr Leu Asn Arg Ser Ala Asp Glu Phe Asn Cys Leu Ser Cys Pro Gly 530 535 540 Ser Met Trp Ser Tyr Lys Asn Asp He Thr Cys Phe Gin Arg Arg Pro 545 550 555 560 Thr Phe Leu Glu Trp Trp His Glu Ala Pro Thr lie Ala Val Ala Leu 565 570 575 Leu Ala Ala Leu Gly Phe Leu Ser Thr Leu Ala He Leu Val He Phe 580 585 590 Trp Arg His Phe Gin Thr Pro He Val Arg Ser Ala Gly Gly Pro Met 595 600 605 Cys Phe Leu Met Leu Thr Leu Leu Leu Val Ala Tyr Met Val Val Pro 610 615 620 Val Tyr Val Gly Pro Pro Lys Val Ser Thr Cys Leu Cys Arg Gin Ala 625 630 635 640 Leu Phe Pro Leu Cys Phe Thr Xie Cys He Ser Cys He Ala Val Arg 645 650 655 Ser Phe Gin lie Val Cys Ala Phe Lys Met Ala Ser Arg Phe Pro Arg 660 665 670 Ala Tyr Ser Tyr Trp Val Arg Tyr Gin Gly Pro Tyr Val Ser Met Ala 675 680 685 Phe He Thr Val Leu Lys Met Val He Val Val He Gly Met Leu Ala 690 695 700 Thr Gly Leu Ser Pro Thr Thr Arg Thr Asp Pro Asp Asp Pro Lys lie 705 710 715 720 Thr Xie Val Ser Cys Asn Pro Asn Tyr Arg Asn Ser Leu Leu Phe Asn 725 730 735 Thr Ser Leu Asp Leu Leu Leu Ser Val Val Gly Phe Ser Phe Ala Tyr 740 745 750 Met Gly Lys Glu Leu Pro Thr Asn Tyr Asn Glu Ala Lys Phe lie Thr 755 760 765 170 173,552/1

Leu Ser 770 Met Thr Phe Tyr Phe 775 Thr Ser Ser Val Ser 780 Leu Cys Thr Phe Met Ser Ala Tyr Ser Gly Val Leu Val Thr lie Val Asp Leu Leu Val 785 790 795 800 Thr Val Leu Asn Leu Leu Ala lie Ser Leu Gly Tyr Phe Gly Pro Lys 805 810 815 Cys Tyr Met Xie Leu Phe Tyr Pro Glu Arg Asn Thr Pro Ala Tyr Phe 820 825 830 Asn Ser Met lie Gin Gly Tyr Thr Met Arg Arg Asp 835 840 &lt;210&gt; 18 &lt;211&gt; 855

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 18 Met Leu Gly Pro Ala Val Leu Gly Leu Ser Leu Trp Ala Leu Leu His 1 5 10 15 Pro Gly Thr Gly Ala Pro Leu Cys Leu Ser Gin Gin Leu Arg Met Lys 20 25 30 Gly Asp Tyr Val Leu Gly Gly Leu Phe Pro Leu Gly Glu Ala Glu Glu 35 40 45 Ala Gly Leu Arg Ser Arg Thr Arg Pro Ser Ser Pro Val Cys Thr Arg 50 55 60 Phe Ser Ser Asn Gly Leu Leu Trp Ala Leu Ala Met Lys Met Ala Val 65 70 75 80 Glu Glu lie Asn Asn Lys Ser Asp Leu Leu Pro Gly Leu Arg Leu Gly 85 90 95 Tyr Asp Leu Phe Asp Thr Cys Ser Glu Pro Val Val Ala Met Lys Pro 100 105 110 Ser Leu Met Phe Leu Ala Lys Ala Gly Ser Arg Asp lie Ala Ala Tyr 115 120 125 Cys Asn Tyr Thr Gin Tyr Gin Pro Arg Val Leu Ala Val lie Gly Pro 130 135 140 His Ser Ser Glu Leu Ala Met Val Thr Gly Lys Phe Phe Ser Phe Phe 145 150 155 160 Leu Met Pro Gin Val Ser Tyr Gly Ala Ser Met Glu Leu Leu Ser Ala 165 170 175 Arg Glu Thr Phe Pro Ser Phe Phe Arg Thr Val Pro Ser Asp Arg Val 180 185 190 Gin Leu Thr Ala Ala Ala Glu Leu Leu Gin Glu Phe Gly Trp Asn Trp 195 200 205 Val Ala Ala Leu Gly Ser Asp Asp Glu Tyr Gly Arg Gin Gly Leu Ser 210 215 220 lie Phe Ser Ala Leu Ala Ala Ala Arg Gly lie Cys lie Ala His Glu 225 230 235 240 Gly Leu Val Pro Leu Pro Arg Ala Asp Asp Ser Arg Leu Gly Lys Val 245 250 255 Gin Asp Val Leu His Gin Val Asn Gin Ser Ser Val Gin Val Val Leu 260 265 270 Leu Phe Ala Ser Val His Ala Ala His Ala Leu Phe Asn Tyr Ser lie 275 280 285 Ser Ser Arg Leu Ser Pro Lys Val Trp Val Ala Ser Glu Ala Trp Leu 290 295 300 Thr Ser Asp Leu Val Met Gly Leu Pro Gly Met Ala Gin Met Gly Thr 305 310 315 320 171 173,552/1

Val Leu Gly Phe Leu 325 Gin Arg Gly Ala Gin 330 Leu His Glu Phe Pro 335 Gin Tyr Val Lys Thr His Leu Ala Leu Ala Thr Asp Pro Ala Phe Cys Ser 340 345 350 Ala Leu Gly Glu Arg Glu Gin Gly Leu Glu Glu Asp Val Val Gly Gin 355 360 365 Arg Cys Pro Gin Cys Asp Cys He Thr Leu Gin Asn Val Ser Ala Gly 370 375 380 Leu Asn His His Gin Thr Phe Ser Val Tyr Ala Ala Val Tyr Ser Val 385 390 395 400 Ala Gin Ala Leu His Asn Thr Leu Gin Cys Asn Ala Ser Gly Cys Pro 405 410 415 Ala Gin Asp Pro Val Lys Pro Trp Gin Leu Leu Glu Asn Met Tyr Asn 420 425 430 Leu Thr Phe His Val Gly Gly Leu Pro Leu Arg Phe Asp Ser Ser Gly 435 440 445 Asn Val Asp Met Glu Tyr Asp Leu Lys Leu Trp Val Trp Gin Gly Ser 450 455 460 Val Pro Arg Leu His Asp Val Gly Arg Phe Asn Gly Ser Leu Arg Thr 465 470 475 480 Glu Arg Leu Lys He Arg Trp His Thr Ser Asp Asn Gin Lys Pro Val 485 490 495 Ser Arg Cys Ser Arg Gin Cys Gin Glu Gly Gin Val Arg Arg Val Lys 500 505 510 Gly Phe His Ser Cys Cys Tyr Asp Cys Val Asp Cys Glu Ala Gly Ser 515 520 525 Tyr Arg Gin Asn Pro Asp Asp He Ala Cys Thr Phe Cys Gly Gin Asp 530 535 540 Glu Trp Ser Pro Glu Arg Ser Thr Arg Cys Phe Arg Arg Arg Ser Arg 545 550 555 560 Phe Leu Glu Leu Ala Trp Gly Glu Pro Ala Val Leu Ser Leu Leu Leu 565 570 575 Leu Leu Cys Leu Val Leu Gly Leu Thr Leu Ala Ala Leu Gly Leu Phe 580 585 590 Val His Tyr Trp Asp Ser Pro Leu Val Gin Ala Ser Gly Gly Ser Leu 595 600 605 Phe Cys Phe Gly Leu lie Cys Leu Gly Leu Phe Cys Leu Ser Val Leu 610 615 620 Leu Phe Pro Gly Arg Pro Arg Ser Ala Ser Cys Leu Ala Gin Gin Pro 625 630 635 640 Met Ala His Leu Pro Leu Thr Gly Cys Leu Ser Thr Leu Phe Leu Gin 645 650 655 Ala Ala Glu lie Phe Val Glu Ser Glu Leu Pro Leu Ser Trp Ala Asn 660 665 670 Trp Leu Cys Ser Tyr Leu Arg Gly Pro Trp Ala Trp Leu Val Val Leu 675 680 685 Leu Ala Thr Leu Val Glu Ala Ala Leu Cys Ala Trp Tyr Leu Met Ala 690 695 700 Phe Pro Pro Glu Val Val Thr Asp Trp Gin Val Leu Pro Thr Glu Val 705 710 715 720 Leu Glu His Cys Arg Met Arg Ser Trp Val Ser Leu Gly Leu Val His 725 730 735 lie Thr Asn Ala Val Leu Ala Phe Leu Cys Phe Leu Gly Thr Phe Leu 740 745 750 Val Gin Ser Gin Pro Gly Arg Tyr Asn Arg Ala Arg Gly Leu Thr Phe 755 760 765 Ala Met Leu Ala Tyr Phe He lie Trp Val Ser Phe Val Pro Leu Leu 770 775 780 Ala Asn Val Gin Val Ala Tyr Gin Pro Ala Val Gin Met Gly Ala He 785 790 795 800 172 173,552/1

Leu Phe Cys Ala Leu 805 Gly lie Leu Ala Thr 810 Phe His Leu Pro Lys 815 Cys Tyr Val Leu Leu Trp Leu Pro Glu Leu Asn Thr Gin Glu Phe Phe Leu 820 825 830 Gly Arg Ser Pro Lys Glu Ala Ser Asp Gly Asn Ser Gly Ser Ser Glu 835 840 845 Ala Thr Arg Gly His Ser Glu 850 855 &lt;210&gt; 19 &lt;211&gt; 859

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 19

Met Pro Gly Leu Ala lie Leu Gly Leu Ser Leu Ala Ala Phe Leu Glu 1 5 10 15 Leu Gly Met Gly Ser Ser Leu Cys Leu Ser Gin Gin Phe Lys Ala Gin 20 25 30 Gly Asp Tyr lie Leu Gly Gly Leu Phe Pro Leu Gly Thr Thr Glu Glu 35 40 45 Ala Thr Leu Asn Gin Arg Thr Gin Pro Asn Gly lie Leu Cys Thr Arg 50 55 60 Phe Ser Pro Leu Gly Leu Phe Leu Ala Met Ala Met Lys Met Ala Val 65 70 75 80 Glu Glu He Asn Asn Gly Ser Ala Leu Leu Pro Gly Leu Arg Leu Gly 85 90 95 Tyr Asp Leu Phe Asp Thr Cys Ser Glu Pro Val Val Thr Met Lys Pro 100 105 110 Ser Leu Met Phe Met Ala Lys Val Gly Ser Gin Ser He Ala Ala Tyr 115 120 125 Cys Asn Tyr Thr Gin Tyr Gin Pro Arg Val Leu Ala Val He Gly Pro 130 135 140 His Ser Ser Glu Leu Ala Leu lie Thr Gly Lys Phe Phe Ser Phe Phe 145 150 155 160 Leu Met Pro Gin Val Ser Tyr Ser Ala Ser Met Asp Arg Leu Ser Asp 165 170 175 Arg Glu Thr Phe Pro Ser Phe Phe Arg Thr Val Pro Ser Asp Arg Val 180 185 190 Gin Leu Gin Ala Val Val Thr Leu Leu Gin Asn Phe Ser Trp Asn Trp 195 200 205 Val Ala Ala Leu Gly Ser Asp Asp Asp Tyr Gly Arg Glu Gly Leu Ser 210 215 220 He Phe Ser Gly Leu Ala Asn Ser Arg Gly He Cys lie Ala His Glu 225 230 235 240 Gly Leu Val Pro Gin His Asp Thr Ser Gly Gin Gin Leu Gly Lys Val 245 250 255 Val Asp Val Leu Arg Gin Val Asn Gin Ser Lys Val Gin Val Val Val 260 265 270 Leu Phe Ala Ser Ala Arg Ala Val Tyr Ser Leu Phe Ser Tyr Ser lie 275 280 285 Leu His Asp Leu Ser Pro Lys Val Trp Val Ala Ser Glu Ser Trp Leu 290 295 300 Thr Ser Asp Leu Val Met Thr Leu Pro Asn He Ala Arg Val Gly Thr 305 310 315 320 Val Leu Gly Phe Leu Gin Arg Gly Ala Leu Leu Pro Glu Phe Ser His 325 330 335 173 173,552/1

Tyr Val Glu Thr 340 Arg Leu Ala Leu Ala Ala Asp Pro Thr 345 Phe 350 Cys Ala Ser Leu Lys Ala Glu Leu Asp Leu Glu Glu Arg Val Met Gly Pro Arg 355 360 365 Cys Ser Gin Cys Asp Tyr Ile Met Leu Gin Asn Leu Ser Ser Gly Leu 370 375 380 Met Gin Asn Leu Ser Ala Gly Gin Leu His His Gin Ile Phe Ala Thr 385 390 395 400 Tyr Ala Ala Val Tyr Ser Val Ala Gin Ala Leu His Asn Thr Leu Gin 405 410 415 Cys Asn Val Ser His Cys His Thr Ser Glu Pro Val Gin Pro Trp Gin 420 425 430 Leu Leu Glu Asn Met Tyr Asn Met Ser Phe Arg Ala Arg Asp Leu Thr 435 440 445 Leu Gin Phe Asp Ala Lys Gly Ser Val Asp Met Glu Tyr Asp Leu Lys 450 455 460 Met Trp Val Trp Gin Ser Pro Thr Pro Val Leu His Thr Val Gly Thr 465 470 475 480 Phe Asn Gly Thr Leu Gin Leu Gin His Ser Lys Met Tyr Trp Pro Gly 485 490 495 Asn Gin Val Pro Val Ser Gin Cys Set Arg Gin Cys Lys Asp Gly Gin 500 505 510 Val Arg Arg Val Lys Gly Phe His Ser Cys Cys Tyr Asp Cys Val Asp 515 520 525 Cys Lys Ala Gly Ser Tyr Arg Lys His Pro Asp Asp Phe Thr Cys Thr 530 535 540 Pro Cys Gly Lys Asp Gin Trp Ser Pro Glu Lys Ser Thr Thr Cys Leu 545 550 555 560 Pro Arg Arg Pro Lys Phe Leu Glu Leu Ala Trp Gly Glu Pro Ala Val 565 570 575 Leu Leu Leu Leu Leu Leu Leu Ser Leu Ala Leu Gly Leu Val Leu Ala 580 585 590 Ala Leu Gly Leu Phe Val His His Arg Asp Ser Pro Leu Val Gin Ala 595 600 605 Ser Gly Gly Pro Leu Ala Cys Phe Gly Leu Val Cys Leu Gly Leu Val 610 615 620 Cys Leu Ser Val Leu Leu Phe Pro Gly Gin Pro Ser Pro Ala Arg Cys 625 630 635 640 Leu Ala Gin Gin Pro Leu Ser His Leu Pro Leu Thr Gly Cys Leu Ser 645 650 655 Thr Leu Phe Leu Gin Ala Ala Glu Xie Phe Val Glu Ser Glu Leu Pro 660 665 670 Leu Ser Trp Ala Asp Arg Leu Ser Gly Cys Leu Arg Gly Pro Trp Ala 675 680 685 Trp Leu Val Val Leu Leu Ala Met Leu Val Glu Val Ala Leu Cys Thr 690 695 700 Trp Tyr Leu Val Ala Phe Pro Pro Glu Val Val Thr Asp Trp His Met 705 710 715 720 Leu Pro Thr Glu Ala Leu Val His Cys Arg Thr Arg Ser Trp Val Ser 725 730 735 Phe Gly Leu Ala His Ala Thr Asn Ala Thr Leu Ala Phe Leu Cys Phe 740 745 750 Leu Gly Thr Phe Leu Val Arg Ser Gin Pro Gly Arg Tyr Asn Arg Ala 755 760 765 Arg Gly Leu Thr Phe Ala Met Leu Ala Tyr Phe Ile Thr Trp Val Ser 770 775 780 Phe Val Pro Leu Leu Ala Asn Val Gin Val Val Leu Arg Pro Ala Val 785 790 795 800 Gin Met Gly Ala Leu Leu Leu Cys Val Leu Gly Ile Leu Ala Ala Phe 805 810 815 174 173,552/1

His Leu Pro Arg 820 Cys Tyr Leu Leu Met Arg Gin Pro Gly Leu Asn Thr 825 830 Pro Glu Phe Phe Leu Gly Gly Gly Pro Gly Asp Ala Gin Gly Gin Asn 835 840 845 Asp Gly Asn Thr Gly Asn Gin Gly Lys His Glu 850 855 &lt;210&gt; 20 &lt;211&gt; 841

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 20 Met Leu Leu Cys Thr Ala Arg Leu Val Gly Leu Gin Leu Leu He Ser 1 5 10 15 Cys Cys Trp Ala Phe Ala Cys His Ser Thr Glu Ser Ser Pro Asp Phe 20 25 30 Thr Leu Pro Gly Asp Tyr Leu Leu Ala Gly Leu Phe Pro Leu His Ser 35 40 45 Gly Cys Leu Gin Val Arg His Arg Pro Glu Val Thr Leu Cys Asp Arg 50 55 60 Ser Cys Ser Phe Asn Glu His Gly Tyr His Leu Phe Gin Ala Met Arg 65 70 75 80 Leu Gly Val Glu Glu He Asn Asn Ser Thr Ala Leu Leu Pro Asn He 85 90 95 Thr Leu Gly Tyr Gin Leu Tyr Asp Val Cys Ser Asp Ser Ala Asn Val 100 105 110 Tyr Ala Thr Leu Arg Val Leu Ser Leu Pro Gly Gin His His He Glu 115 120 125 Leu Gin Gly Asp Leu Leu His Tyr Ser Pro Thr Val Leu Ala Val He 130 135 140 Gly Pro Asp Ser Thr Asn Arg Ala Ala Thr Thr Ala Ala Leu Leu Ser 145 150 155 160 Pro Phe Leu Val Pro Met lie Ser Tyr Ala Ala Ser Ser Glu Thr Leu 165 170 175 Ser Val Lys Arg Gin Tyr Pro Ser Phe Leu Arg Thr lie Pro Asn Asp 180 185 190 Lys Tyr Gin Val Glu Thr Met Val Leu Leu Leu Gin Lys Phe Gly Trp 195 200 205 Thr Trp He Ser Leu Val Gly Ser Ser Asp Asp Tyr Gly Gin Leu Gly 210 215 220 Val Gin Ala Leu Glu Asn Gin Ala Thr Gly Gin Gly He Cys He Ala 225 230 235 240 Phe Lys Asp lie Met Pro Phe Ser Ala Gin Val Gly Asp Glu Arg Met 245 250 255 Gin Cys Leu Met Arg His Leu Ala Gin Ala Gly Ala Thr Val Val Val 260 265 270 Val Phe Ser Ser Arg Gin Leu Ala Arg Val Phe Phe Glu Ser Val Val 275 280 285 Leu Thr Asn Leu Thr Gly Lys Val Trp Val Ala Ser Glu Ala Trp Ala 290 295 300 Leu Ser Arg His He Thr Gly Val Pro Gly lie Gin Arg He Gly Met 305 310 315 320 Val Leu Gly Val Ala He Gin Lys Arg Ala Val Pro Gly Leu Lys Ala 325 330 335 Phe Glu Glu Ala Tyr Ala Arg Ala Asp Lys Lys Ala Pro Arg Pro Cys 340 345 350 175 173,552/1

His Lys Gly 355 Ser Trp Cys Ser Ser 360 Asn Gin Leu Cys Arg 365 Glu Cys Gin Ala Phe Met Ala His Thr Met Pro Lys Leu Lys Ala Phe Ser Met Ser 370 375 380 Ser Ala Tyr Asn Ala Tyr Arg Ala Val Tyr Ala Val Ala His Gly Leu 385 390 395 400 His Gin Leu Leu Gly Cys Ala Ser Gly Ala Cys Ser Arg Gly Arg Val 405 410 415 Tyr Pro Trp Gin Leu Leu Glu Gin He His Lys Val His Phe Leu Leu 420 425 430 His Lys Asp Thr Val Ala Phe Asn Asp Asn Arg Asp Pro Leu Ser Ser 435 440 445 Tyr Asn He lie Ala Trp Asp Trp Asn Gly Pro Lys Trp Thr Phe Thr 450 455 460 Val Leu Gly Ser Ser Thr Trp Ser Pro Val Gin Leu Asn He Asn Glu 465 470 475 480 Thr Lys He Gin Trp His Gly Lys Asp Asn Gin Val Pro Lys Ser Val 485 490 495 Cys Ser Ser Asp Cys Leu Glu Gly His Gin Arg Val Val Thr Gly Phe 500 505 510 His His Cys Cys Phe Glu Cys Val Pro Cys Gly Ala Gly Thr Phe Leu 515 520 525 Asn Lys Ser Asp Leu Tyr Arg Cys Gin Pro Cys Gly Lys Glu Glu Trp 530 535 540 Ala Pro Glu Gly Ser Gin Thr Cys Phe Pro Arg Thr Val Val Phe Leu 545 550 555 560 Glu Trp His Glu Pro He Ser Leu Val Leu He Ala Ala Asn Thr Leu 565 570 575 Leu Leu Leu Leu Leu Val Gly Thr Ala Gly Leu Phe Ala Trp His Phe 580 585 590 His Thr Pro Val Val Arg Ser Ala Gly Gly Arg Leu Cys Phe Leu Met 595 600 605 Leu Gly Ser Leu Val Ala Gly Ser Cys Ser Phe Tyr Ser Phe Phe Gly 610 615 620 Glu Pro Thr Val Pro Ala Cys Leu Leu Arg Gin Pro Leu Phe Ser Leu 625 630 635 640 Gly Phe Ala He Phe Leu Ser Cys Leu Thr He Arg Ser Phe Gin Leu 645 650 655 Val lie He Phe Lys Phe Ser Thr Lys Val Pro Thr Phe Tyr Arg Thr 660 665 670 Trp Ala Gin Asn His Gly Ala Gly Leu Phe Val He Val Ser Ser Thr 675 680 685 Val His Leu Leu He Cys Leu Thr Trp Leu Val Met Trp Thr Pro Arg 690 695 700 Pro Thr Arg Glu Tyr Gin Arg Phe Pro His Leu Val He Leu Glu Cys 705 710 715 720 Thr Glu Val Asn Ser Val Gly Phe Leu Leu Ala Phe Thr His Asn lie 725 730 735 Leu Leu Ser He Ser Thr Phe Val Cys Ser Tyr Leu Gly Lys Glu Leu 740 745 750 Pro Glu Asn Tyr Asn Glu Ala Lys Cys Val Thr Phe Ser Leu Leu Leu 755 760 765 Asn Phe Val Ser Trp He Ala Phe Phe Thr Met Ala Ser lie Tyr Gin 770 775 780 Gly Ser Tyr Leu Pro Ala Val Asn Val Leu Ala Gly Leu Thr Thr Leu 785 790 795 800 Ser Gly Gly Phe Ser Gly Tyr Phe Leu Pro Lys Cys Tyr Val He Leu 805 810 815 Cys Arg Pro Glu Leu Asn Asn Thr Glu His Phe Gin Ala Ser He Gin 820 825 830 176 173,552/1

Asp Tyr Thr Arg Arg Cys Gly Thr Thr 835 840 &lt;210&gt; 21 &lt;211&gt; 840

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 21

Met 1 Leu Phe Trp Ala 5 Ala His Leu Leu Leu 10 Ser Leu Gin Leu Val 15 Tyr Cys Trp Ala Phe Ser Cys Gin Arg Thr Glu Ser Ser Pro Gly Phe Ser 20 25 30 Leu Pro Gly Asp Phe Leu Leu Ala Gly Leu Phe Ser Leu His Gly Asp 35 40 45 Cys Leu Gin Val Arg His Arg Pro Leu Val Thr Ser Cys Asp Arg Pro 50 55 60 Asp Ser Phe Asn Gly His Gly Tyr His Leu Phe Gin Ala Met Arg Phe 65 70 75 80 Thr Val Glu Glu Ile Asn Asn Ser Ser Ala Leu Leu Pro Asn Ile Thr 85 90 95 Leu Gly Tyr Glu Leu Tyr Asp Val Cys Ser Glu Ser Ala Asn Val Tyr 100 105 110 Ala Thr Leu Arg Val Leu Ala Leu Gin Gly Pro Arg His Ile Glu Ile 115 120 125 Gin Lys Asp Leu Arg Asn His Ser Ser Lys Val Val Ala Phe Ile Gly 130 135 140 Pro Asp Asn Thr Asp His Ala Val Thr Thr Ala Ala Leu Leu Gly Pro 145 150 155 160 Phe Leu Met Pro Leu Val Ser Tyr Glu Ala Ser Ser Val Val Leu Ser 165 170 175 Ala Lys Arg Lys Phe Pro Ser Phe Leu Arg Thr Val Pro Ser Asp Arg 180 185 190 His Gin Val Glu Val Met Val Gin Leu Leu Gin Ser Phe Gly Trp Val 195 200 205 Trp Ile Ser Leu Ile Gly Ser Tyr Gly Asp Tyr Gly Gin Leu Gly Val 210 215 220 Gin Ala Leu Glu Glu Leu Ala Val Pro Arg Gly lie Cys Val Ala Phe 225 230 235 240 Lys Asp Ile Val Pro Phe Ser Ala Arg Val Gly Asp Pro Arg Met Gin 245 250 255 Ser Met Met Gin His Leu Ala Gin Ala Arg Thr Thr Val Val Val Val 260 265 270 Phe Ser Asn Arg His Leu Ala Arg Val Phe Phe Arg Ser Val Val Leu 275 280 285 Ala Asn Leu Thr Gly Lys Val Trp Val Ala Ser Glu Asp Trp Ala Ile 290 295 300 Ser Thr Tyr lie Thr Ser Val Thr Gly Ile Gin Gly Ile Gly Thr Val 305 310 315 320 Leu Gly Val Ala Val Gin Gin Arg Gin Val Pro Gly Leu Lys Glu Phe 325 330 335 Glu Glu Ser Tyr Val Arg Ala Val Thr Ala Ala Pro Ser Ala Cys Pro 340 345 350 Glu Gly Ser Trp Cys Ser Thr Asn Gin Leu Cys Arg Glu Cys His Thr 355 360 365 Phe Thr Thr Arg Asn Met Pro Thr Leu Gly Ala Phe Ser Met Ser Ala 370 375 380 177 173,552/1

Ala Tyr Arg Val Tyr Glu Ala Val Tyr Ala Val Ala His Gly Leu His 385 390 395 400 Gin Leu Leu Gly Cys Thr Ser Glu Ile Cys Ser Arg Gly Pro Val Tyr 405 410 415 Pro Trp Gin Leu Leu Gin Gin Ile Tyr Lys Val Asn Phe Leu Leu His 420 425 430 Glu Asn Thr Val Ala Phe Asp Asp Asn Gly Asp Thr Leu Gly Tyr Tyr 435 440 445 Asp Ile Ile Ala Trp Asp Trp Asn Gly Pro Glu Trp Thr Phe Glu Ile 450 455 460 Ile Gly Ser Ala Ser Leu Ser Pro Val His Leu Asp Ile Asn Lys Thr 465 470 475 480 Lys Ile Gin Trp His Gly Lys Asn Asn Gin Val Pro Val Ser Val Cys 485 490 495 Thr Thr Asp Cys Leu Ala Gly His His Arg Val Val Val Gly Ser His 500 505 510 His Cys Cys Phe Glu Cys Val Pro Cys Glu Ala Gly Thr Phe Leu Asn 515 520 525 Met Ser Glu Leu His Ile Cys Gin Pro Cys Gly Thr Glu Glu Trp Ala 530 535 540 Pro Lys Glu Ser Thr Thr Cys Phe Pro Arg Thr Val Glu Phe Leu Glu 545 550 555 560 Leu Arg Glu His Thr Ser Trp Val Leu Leu Ala Ala Asn Thr Leu Leu 565 570 575 Leu Leu Leu Leu Leu Gly Thr Ala Gly Leu Phe Ala Trp His Leu Asp 580 585 590 Thr Pro Val Val Arg Ser Ala Gly Gly Arg Leu Cys Phe Leu Met Leu 595 600 605 Gly Ser Leu Ala Ala Gly Ser Gly Ser Leu Tyr Gly Phe Phe Gly Glu 610 615 620 Pro Thr Arg Pro Ala Cys Leu Leu Arg Gin Ala Leu Phe Ala Leu Gly 625 630 635 640 Phe Thr Ile Phe Leu Ser Cys Leu Thr Val Arg Ser Phe Gin Leu Ile 645 650 655 lie Ile Phe Lys Phe Ser Thr Lys Val Pro Thr Phe Tyr His Ala Trp 660 665 670 Val Gin Asn His Gly Ala Gly Leu Phe Val Met Ile Ser Ser Ala Ala 675 680 685 Gin Leu Leu Ile Cys Leu Thr Trp Leu Val Val Trp Thr Pro Leu Pro 690 695 700 Ala Arg Glu Tyr Gin Arg Phe Pro His Leu Val Met Leu Glu Cys Thr 705 710 715 720 Glu Thr Asn Ser Leu Gly Phe Ile Leu Ala Phe Leu Tyr Asn Gly Leu 725 730 735 Leu Ser lie Ser Ala Phe Ala Cys Ser Tyr Leu Gly Lys Asp Leu Pro 740 745 750 Glu Asn Tyr Asn Glu Ala Lys Cys Val Thr Phe Ser Leu Leu Phe Asn 755 760 765 Phe Val Ser Trp Ile Ala Phe Phe Thr Thr Ala Ser Val Tyr Asp Giy 770 775 780 Lys Tyr Leu Pro Ala Ala Asn Met Met Ala Gly Leu Ser Ser Leu Ser 785 790 795 800 Ser Gly Phe Gly Gly Tyr Phe Leu Pro Lys Cys Tyr Val Ile Leu Cys 805 810 815 Arg Pro Asp Leu Asn Ser Thr Glu His Phe Gin Ala Ser Ile Gin Asp 820 825 830 Tyr Thr Arg Arg Cys Gly Ser Thr 835 840 &lt;210&gt; 22 &lt;211&gt; 838 178

<img img-format="tif" img-content="drawing" file="IL173552AD000291.tif" id="idf0091" />

173,552/1

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 22

Met Gly Pro Arg Ala Lys Thr He Cys Ser Leu Phe Phe Leu Leu Trp 1 5 10 15 Val Leu Ala Glu Pro Ala Glu Asn Ser Asp Phe Tyr Leu Pro Gly Asp 20 25 30 Tyr Leu Leu Gly Gly Leu Phe Ser Leu His Ala Asn Met Lys Gly He 35 40 45 Val His Leu Asn Phe Leu Gin Val Pro Met Cys Lys Glu Tyr Glu Val 50 55 60 Lys Val lie Gly Tyr Asn Leu Met Gin Ala Met Arg Phe Ala Val Glu 65 70 75 80 Glu lie Asn Asn Asp Ser Ser Leu Leu Pro Gly Val Leu Leu Gly Tyr 85 90 95 Glu He Val Asp Val Cys Tyr He Ser Asn Asn Val Gin Pro Val Leu 100 105 110 Tyr Phe Leu Ala His Glu Asp Asn Leu Leu Pro lie Gin Glu Asp Tyr 115 120 125 Ser Asn Tyr He Ser Arg Val Val Ala Val lie Gly Pro Asp Asn Ser 130 135 140 Glu Ser Val Met Thr Val Ala Asn Phe Leu Ser Leu Phe Leu Leu Pro 145 150 155 160 Gin He Thr Tyr Ser Ala lie Ser Asp Glu Leu Arg Asp Lys Val Arg 165 170 175 Phe Pro Ala Leu Leu Arg Thr Thr Pro Ser Ala Asp His His Val Glu 180 185 190 Ala Met Val Gin Leu Met Leu His Phe Arg Trp Asn Trp He He Val 195 200 205 Leu Val Ser Ser Asp Thr Tyr Gly Arg Asp Asn Gly Gin Leu Leu Gly 210 215 220 Glu Arg Val Ala Arg Arg Asp lie Cys He Ala Phe Gin Glu Thr Leu 225 230 235 240 Pro Thr Leu Gin Pro Asn Gin Asn Met Thr Ser Glu Glu Arg Gin Arg 245 250 255 Leu Val Thr He Val Asp Lys Leu Gin Gin Ser Thr Ala Arg Val Val 260 265 270 Val Val Phe Ser Pro Asp Leu Thr Leu Tyr His Phe Phe Asn Glu Val 275 280 285 Leu Arg Gin Asn Phe Thr Gly Ala Val Trp lie Ala Ser Glu Ser Trp 290 295 300 Ala lie Asp Pro Val Leu His Asn Leu Thr Glu Leu Gly His Leu Gly 305 310 315 320 Thr Phe Leu Gly He Thr He Gin Ser Val Pro He Pro Gly Phe Ser 325 330 335 Glu Phe Arg Glu Trp Gly Pro Gin Ala Gly Pro Pro Pro Leu Ser Arg 340 345 350 Thr Ser Gin Ser Tyr Thr Cys Asn Gin Glu Cys Asp Asn Cys Leu Asn 355 360 365 Ala Thr Leu Ser Phe Asn Thr lie Leu Arg Leu Ser Gly Glu Arg Val 370 375 380 Val Tyr Ser Val Tyr Ser Ala Val Tyr Ala Val Ala His Ala Leu His 385 390 395 400 Ser Leu Leu Gly Cys Asp Lys Ser Thr Cys Thr Lys Arg Val Val Tyr 405 410 415 179 173,552/1

Pro Trp Gin Leu 420 Leu Glu Glu He Trp 425 Lys Val Asn Phe Thr 430 Leu Leu Asp His Gin lie Phe Phe Asp Pro Gin Gly Asp Val Ala Leu His Leu 435 440 445 Glu He Val Gin Trp Gin Trp Asp Arg Ser Gin Asn Pro Phe Gin Ser 450 455 460 Val Ala Ser Tyr Tyr Pro Leu Gin Arg Gin Leu Lys Asn He Gin Asp 465 470 475 480 lie Ser Trp His Thr Val Asn Asn Thr He Pro Met Ser Met Cys Ser 485 490 495 Lys Arg Cys Gin Ser Gly Gin Lys Lys Lys Pro Val Gly He His Val 500 505 510 Cys Cys Phe Glu Cys He Asp Cys Leu Pro Gly Thr Phe Leu Asn His 515 520 525 Thr Glu Asp Glu Tyr Glu Cys Gin Ala Cys Pro Asn Asn Glu Trp Ser 530 535 540 Tyr Gin Ser Glu Thr Ser Cys Phe Lys Arg Gin Leu Val Phe Leu Glu 545 550 555 560 His Glu Val Pro Thr lie Val Val Ala He Leu Ala Ala Leu Gly Phe 565 570 575 Phe Ser Thr Leu Ala lie Leu Phe He Phe Trp Arg His Phe Gin Thr 580 585 590 Pro Met Val Arg Ser Ala Gly Gly Pro Met Cys Phe Leu Met Leu Val 595 600 605 Pro Leu Leu Leu Ala Phe Gly Met Val Pro Val Tyr Val Gly Pro Pro 610 615 620 Thr Val Phe Ser Cys Phe Cys Arg Gin Ala Phe Phe Thr Val Cys Phe 625 630 635 640 Ser He Cys Leu Ser Cys He Thr Val Arg Ser Phe Gin He Val Cys 645 650 655 Val Phe Lys Met Ala Arg Arg Leu Pro Ser Ala Tyr Ser Phe Trp Met 660 665 670 Arg Tyr His Gly Pro Tyr Val Phe Val Ala Phe He Thr Ala He Lys 675 680 685 Val Ala Leu Val Val Gly Asn Met Leu Ala Thr Thr lie Asn Pro lie 690 695 700 Gly Arg Thr Asp Pro Asp Asp Pro Asn He Met He Leu Ser Cys His 705 710 715 720 Pro Asn Tyr Arg Asn Gly Leu Leu Phe Asn Thr Ser Met Asp Leu Leu 725 730 735 Leu Ser Val Leu dy Phe Ser Phe Ala Tyr Met Gly Lys Glu Leu Pro 740 745 750 Thr Asn Tyr Asn Glu Ala Lys Phe He Thr Leu Ser Met Thr Phe Ser 755 760 765 Phe Thr Ser Ser He Ser Leu Cys Thr Phe Met Ser Val His Asp Gly 770 775 780 Val Leu Val Thr He Met Asp Leu Leu Val Thr Val Leu Asn Phe Leu 785 790 795 800 Ala He Gly Leu Gly Tyr Phe Gly Pro Lys Cys Tyr Met lie Leu Phe 805 810 815 Tyr Pro Glu Arg Asn Thr Ser Ala Tyr Phe Asn Ser Met lie Gin Gly 820 825 830 Tyr Thr Met Arg Lys Ser 835 &lt;210&gt; 23 &lt;211&gt; 843

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence 180 173,552/1 &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 23 Met Gly Pro Gin Ala Arg Thr Leu Cys Leu Leu Ser Leu Leu Leu His 1 5 10 15 Val Leu Pro Lys Pro Gly Lys Leu Val Glu Asn Ser Asp Phe His Leu 20 25 30 Ala Gly Asp Tyr Leu Leu Gly Gly Leu Phe Thr Leu His Ala Asn Val 35 40 45 Lys Ser He Ser His Leu Ser Tyr Leu Gin Val Pro Lys Cys Asn Glu 50 55 60 Phe Thr Met Lys Val Leu Gly Tyr Asn Leu Met Gin Ala Met Arg Phe 65 70 75 80 Ala Val Glu Glu He Asn Asn Cys Ser Ser Leu Leu Pro Gly Val Leu 85 90 95 Leu Gly Tyr Glu Met Val Asp Val Cys Tyr Leu Ser Asn Asn lie His 100 105 110 Pro Gly Leu Tyr Phe Leu Ala Gin Asp Asp Asp Leu Leu Pro He Leu 115 120 125 Lys Asp Tyr Ser Gin Tyr Met Pro His Val Val Ala Val He Gly Pro 130 135 140 Asp Asn Ser Glu Ser Ala He Thr Val Ser Asn He Leu Ser His Phe 145 150 155 160 Leu lie Pro Gin He Thr Tyr Ser Ala He Ser Asp Lys Leu Arg Asp 165 170 175 Lys Arg His Phe Pro Ser Met Leu Arg Thr Val Pro Ser Ala Thr His 180 185 190 His lie Glu Ala Met Val Gin Leu Met Val His Phe Gin Trp Asn Trp 195 200 205 lie Val Val Leu Val Ser Asp Asp Asp Tyr Gly Arg Glu Asn Ser His 210 215 220 Leu Leu Ser Gin Arg Leu Thr Lys Thr Ser Asp He Cys He Ala Phe 225 230 235 240 Gin Glu Val Leu Pro He Pro Glu Ser Ser Gin Val Met Arg Ser Glu 245 250 255 Glu Gin Arg Gin Leu Asp Asn He Leu Asp Lys Leu Arg Arg Thr Ser 260 265 270 Ala Arg Val Val Val Val Phe Ser Pro Glu Leu Ser Leu Tyr Ser Phe 275 280 285 Phe His Glu Val Leu Arg Trp Asn Phe Thr Gly Phe Val Trp He Ala 290 295 300 Ser Glu Ser Trp Ala lie Asp Pro Val Leu His Asn Leu Thr Glu Leu 305 310 315 320 Arg His Thr Gly Thr Phe Leu Gly Val Thr He Gin Arg Val Ser He 325 330 335 Pro Gly Phe Ser Gin Phe Arg Val Arg Arg Asp Lys Pro Gly Tyr Pro 340 345 350 Val Pro Asn Thr Thr Asn Leu Arg Thr Thr Cys Asn Gin Asp Cys Asp 355 360 365 Ala Cys Leu Asn Thr Thr Lys Ser Phe Asn Asn He Leu He Leu Ser 370 375 380 Gly Glu Arg Val Val Tyr Ser Val Tyr Ser Ala Val Tyr Ala Val Ala 385 390 395 400 His Ala Leu His Arg Leu Leu Gly Cys Asn Arg Val Arg Cys Thr Lys 405 410 415 Gin Lys Val Tyr Pro Trp Gin Leu Leu Arg Glu lie Trp His Val Asn 420 425 430 181 173,552/1

Phe Thr Leu 435 Leu Gly Asn Arg Leu 440 Phe Phe Asp Gin Gin 445 Gly Asp Met Pro Met Leu Leu Asp He He Gin Trp Gin Trp Asp Leu Ser Gin Asn 450 455 460 Pro Phe Gin Ser He Ala Ser Tyr Ser Pro Thr Ser Lys Arg Leu Thr 465 470 475 480 Tyr He Asn Asn Val Ser Trp Tyr Thr Pro Asn Asn Thr Val Pro Val 485 490 495 Ser Met Cys Ser Lys Ser Cys Gin Pro Gly Gin Met Lys Lys Ser Val 500 505 510 Gly Leu His Pro Cys Cys Phe Glu Cys Leu Asp Cys Met Pro Gly Thr 515 520 525 Tyr Leu Asn Arg Ser Ala Asp Glu Phe Asn Cys Leu Ser Cys Pro Gly 530 535 540 Ser Met Trp Ser Tyr Lys Asn Asp Xie Thr Cys Phe Gin Arg Arg Pro 545 550 555 560 Thr Phe Leu Glu Trp His Glu Ala Pro Thr lie Ala Val Ala Leu Leu 565 570 575 Ala Ala Leu Gly Phe Leu Ser Thr Leu Ala He Leu Val He Phe Trp 580 585 590 Arg His Phe Gin Thr Pro He Val Arg Ser Ala Gly Gly Pro Met Cys 595 600 605 Phe Leu Met Leu Thr Leu Leu Leu Val Ala Tyr Met Val Val Pro Val 610 615 620 Tyr Val Gly Pro Pro Lys Val Ser Thr Cys Leu Cys Arg Gin Ala Leu 625 630 635 640 Phe Pro Leu Cys Phe Thr lie Cys He Ser Cys He Ala Val Arg Ser 645 650 655 Phe Gin lie Val Cys Ala Phe Lys Met Ala Ser Arg Phe Pro Arg Ala 660 665 670 Tyr Ser Tyr Trp Val Arg Tyr Gin Gly Pro Tyr Val Ser Met Ala Phe 675 680 685 lie Thr Val Leu Lys Met Val He Val Val lie Gly Met Leu Ala Thr 690 695 700 Gly Leu Ser Pro Thr Thr Arg Thr Asp Pro Asp Asp Pro Lys He Thr 705 710 715 720 He Val Ser Cys Asn Pro Asn Tyr Arg Asn Ser Leu Leu Phe Asn Thr 725 730 735 Ser Leu Asp Leu Leu Leu Ser Val Val Gly Phe Ser Phe Ala Tyr Met 740 745 750 Gly Lys Glu Leu Pro Thr Asn Tyr Asn Glu Ala Lys Phe He Thr Leu 755 760 765 Ser Met Thr Phe Tyr Phe Thr Ser Ser Val Ser Leu Cys Thr Phe Met 770 775 780 Ser Ala Tyr Ser Gly Val Leu Val Thr lie Val Asp Leu Leu Val Thr 785 790 795 800 Val Leu Asn Leu Leu Ala He Ser Leu Gly Tyr Phe Gly Pro Lys Cys 805 810 815 Tyr Met He Leu Phe Tyr Pro Glu Arg Asn Thr Pro Ala Tyr Phe Asn 820 825 830 Ser Met He Gin Gly Tyr Thr Met Arg Arg Asp 835 840 &lt;210&gt; 24 &lt;2Π&gt; 853

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct 182 173,552/1 &lt;400&gt; 24

Met Leu Gly Pro Ala Val Leu Gly Leu Ser Leu Trp Ala Leu Leu His 1 5 10 15 Pro Gly Thr Gly Ala Pro Leu Cys Leu Ser Gin Gin Leu Arg Met Lys 20 25 30 Gly Asp Tyr Val Leu Gly Gly Leu Phe Pro Leu Gly Glu Ala Glu Glu 35 40 45 Ala Gly Leu Arg Ser Arg Thr Arg Pro Ser Ser Pro Val Cys Thr Arg 50 55 60 Phe Ser Ser Asn Gly Leu Leu Trp Ala Leu Ala Met Lys Met Ala Val 65 70 75 80 Glu Glu He Asn Asn Lys Ser Asp Leu Leu Pro Gly Leu Arg Leu Gly 85 90 95 Tyr Asp Leu Phe Asp Thr Cys Ser Glu Pro Val Val Ala Met Lys Pro 100 105 110 Ser Leu Met Phe Leu Ala Lys Ala Gly Ser Arg Asp He Ala Ala Tyr 115 120 125 Cys Asn Tyr Thr Gin Tyr Gin Pro Arg Val Leu Ala Val lie Gly Pro 130 135 140 His Ser Ser Glu Leu Ala Met Val Thr Gly Lys Phe Phe Ser Phe Phe 145 150 155 160 Leu Met Pro Gin Val Ser Tyr Gly Ala Ser Met Glu Leu Leu Ser Ala 165 170 175 Arg Glu Thr Phe Pro Ser Phe Phe Arg Thr Val Pro Ser Asp Arg Val 180 185 190 Gin Leu Thr Ala Ala Ala Glu Leu Leu Gin Glu Phe Gly Trp Asn Trp 195 200 205 Val Ala Ala Leu Gly Ser Asp Asp Glu Tyr Gly Arg Gin Gly Leu Ser 210 215 220 lie Phe Ser Ala Leu Ala Ala Ala Arg Gly lie Cys lie Ala His Glu 225 230 235 240 Gly Leu Val Pro Leu Pro Arg Ala Asp Asp Ser Arg Leu Gly Lys Val 245 250 255 Gin Asp Val Leu His Gin Val Asn Gin Ser Ser Val Gin Val Val Leu 260 265 270 Leu Phe Ala Ser Val His Ala Ala His Ala Leu Phe Asn Tyr Ser lie 275 280 285 Ser Ser Arg Leu Ser Pro Lys Val Trp Val Ala Ser Glu Ala Trp Leu 290 295 300 Thr Ser Asp Leu Val Met Gly Leu Pro Gly Met Ala Gin Met Gly Thr 305 310 315 320 Val Leu Gly Phe Leu Gin Arg Gly Ala Gin Leu His Glu Phe Pro Gin 325 330 335 Tyr Val Lys Thr His Leu Ala Leu Ala Thr Asp Pro Ala Phe Cys Ser 340 345 350 Ala Leu Gly Glu Arg Glu Gin Gly Leu Glu Glu Asp Val Val Gly Gin 355 360 365 Arg Cys Pro Gin Cys Asp Cys He Thr Leu Gin Asn Val Ser Ala Gly 370 375 380 Leu Asn His His Gin Thr Phe Ser Val Tyr Ala Ala Val Tyr Ser Val 385 390 395 400 Ala Gin Ala Leu His Asn Thr Leu Gin Cys Asn Ala Ser Gly Cys Pro 405 410 415 Ala Gin Asp Pro Val Lys Pro Trp Gin Leu Leu Glu Asn Met Tyr Asn 420 425 430 Leu Thr Phe His Val Gly Gly Leu Pro Leu Arg Phe Asp Ser Ser Gly 435 440 445 Asn Val Asp Met Glu Tyr Asp Leu Lys Leu Trp Val Trp Gin Gly Ser 450 455 460 183 173,552/1

Val Pro Arg Leu His Asp Val Gly Arg Phe Asn Gly Ser Leu Arg Thr 465 470 475 480 Glu Arg Leu Lys Ile Arg Trp His Thr Ser Asp Asn Gin Lys Pro Val 485 490 495 Ser Arg Cys Ser Arg Gin Cys Gin Glu Gly Gin Val Arg Arg Val Lys 500 505 510 Gly Phe His Ser Cys Cys Tyr Asp Cys Val Asp Cys Glu Ala Gly Ser 515 520 525 Tyr Arg Gin Asn Pro Asp Asp Ile Ala Cys Thr Phe Cys Gly Gin Asp 530 535 540 Glu Trp Ser Pro Glu Arg Ser Thr Arg Cys Phe Arg Arg Arg Ser Arg 545 550 555 560 Phe Leu Glu Trp Gly Glu Pro Ala Val Leu Ser Leu Leu Leu Leu Leu 565 570 575 Cys Leu Val Leu Gly Leu Thr Leu Ala Ala Leu Gly Leu Phe Val His 580 585 590 Tyr Trp Asp Ser Pro Leu Val Gin Ala Ser Gly Gly Ser Leu Phe Cys 595 600 605 Phe Gly Leu He Cys Leu Gly Leu Phe Cys Leu Ser Val Leu Leu Phe 610 615 620 Pro Gly Arg Pro Arg Ser Ala Ser Cys Leu Ala Gin Gin Pro Met Ala 625 630 635 640 His Leu Pro Leu Thr Gly Cys Leu Ser Thr Leu Phe Leu Gin Ala Ala 645 650 655 Glu Ile Phe Val Glu Ser Glu Leu Pro Leu Ser Trp Ala Asn Trp Leu 660 665 670 Cys Ser Tyr Leu Arg Gly Pro Trp Ala Trp Leu Val Val Leu Leu Ala 675 680 685 Thr Leu Val Glu Ala Ala Leu Cys Ala Trp Tyr Leu Met Ala Phe Pro 690 695 700 Pro Glu Val Val Thr Asp Trp Gin Val Leu Pro Thr Glu Val Leu Glu 705 710 715 720 His Cys Arg Met Arg Ser Trp Val Ser Leu Gly Leu Val His Ile Thr 725 730 735 Asn Ala Val Leu Ala Phe Leu Cys Phe Leu Gly Thr Phe Leu Val Gin 740 745 750 Ser Gin Pro Gly Arg Tyr Asn Arg Ala Arg Gly Leu Thr Phe Ala Met 755 760 765 Leu Ala Tyr Phe Ile Ile Trp Val Ser Phe Val Pro Leu Leu Ala Asn 770 775 780 Val Gin Val Ala Tyr Gin Pro Ala Val Gin Met Gly Ala Ile Leu Phe 785 790 795 800 Cys Ala Leu Gly Ile Leu Ala Thr Phe His Leu Pro Lys Cys Tyr Val 805 810 815 Leu Leu Trp Leu Pro Glu Leu Asn Thr Gin Glu Phe Phe Leu Gly Arg 820 825 830 Ser Pro Lys Glu Ala Ser Asp Gly Asn Ser Gly Ser Ser Glu Ala Thr 835 840 845 Arg Gly His Ser Glu 850 &lt;210&gt; 25 &lt;211&gt; 857

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct 184 173,552/1 &lt;400&gt; 25 Met Pro Gly Leu Ala lie Leu Gly Leu Ser Leu Ala Ala Phe Leu Glu 1 5 10 15 Leu Gly Met Gly Ser Ser Leu Cys Leu Ser Gin Gin Phe Lys Ala Gin 20 25 30 Gly Asp Tyr lie Leu Gly Gly Leu Phe Pro Leu Gly Thr Thr Glu Glu 35 40 45 Ala Thr Leu Asn Gin Arg Thr Gin Pro Asn Gly lie Leu Cys Thr Arg 50 55 60 Phe Ser Pro Leu Gly Leu Phe Leu Ala Met Ala Met Lys Met Ala Val 65 70 75 80 Glu Glu Xie Asn Asn Gly Ser Ala Leu Leu Pro Gly Leu Arg Leu Gly 85 90 95 Tyr Asp Leu Phe Asp Thr Cys Ser Glu Pro Val Val Thr Met Lys Pro 100 105 110 Ser Leu Met Phe Met Ala Lys Val Gly Ser Gin Ser lie Ala Ala Tyr 115 120 125 Cys Asn Tyr Thr Gin Tyr Gin Pro Arg Val Leu Ala Val lie Gly Pro 130 135 140 His Ser Ser Glu Leu Ala Leu lie Thr Gly Lys Phe Phe Ser Phe Phe 145 150 155 160 Leu Met Pro Gin Val Ser Tyr Ser Ala Ser Met Asp Arg Leu Ser Asp 165 170 175 Arg Glu Thr Phe Pro Ser Phe Phe Arg Thr Val Pro Ser Asp Arg Val 180 185 190 Gin Leu Gin Ala Val Val Thr Leu Leu Gin Asn Phe Ser Trp Asn Trp 195 200 205 Val Ala Ala Leu Gly Ser Asp Asp Asp Tyr Gly Arg Glu Gly Leu Ser 210 215 220 Xie Phe Ser Gly Leu Ala Asn Ser Arg Gly lie Cys lie Ala His Glu 225 230 235 240 Gly Leu Val Pro Gin His Asp Thr Ser Gly Gin Gin Leu Gly Lys Val 245 250 255 Val Asp Val Leu Arg Gin Val Asn Gin Ser Lys Val Gin Val Val Val 260 265 270 Leu Phe Ala Ser Ala Arg Ala Val Tyr Ser Leu Phe Ser Tyr Ser lie 275 280 285 Leu His Asp Leu Ser Pro Lys Val Trp Val Ala Ser Glu Ser Trp Leu 290 295 300 Thr Ser Asp Leu Val Met Thr Leu Pro Asn lie Ala Arg Val Gly Thr 305 310 315 320 Val Leu Gly Phe Leu Gin Arg Gly Ala Leu Leu Pro Glu Phe Ser His 325 330 335 Tyr Val Glu Thr Arg Leu Ala Leu Ala Ala Asp Pro Thr Phe Cys Ala 340 345 350 Ser Leu Lys Ala Glu Leu Asp Leu Glu Glu Arg Val Met Gly Pro Arg 355 360 365 Cys Ser Gin Cys Asp Tyr lie Met Leu Gin Asn Leu Ser Ser Gly Leu 370 375 380 Met Gin Asn Leu Ser Ala Gly Gin Leu His His Gin lie Phe Ala Thr 385 390 395 400 Tyr Ala Ala Val Tyr Ser Val Ala Gin Ala Leu His Asn Thr Leu Gin 405 410 415 Cys Asn Val Ser His Cys His Thr Ser Glu Pro Val Gin Pro Trp Gin 420 425 430 Leu Leu Glu Asn Met Tyr Asn Met Ser Phe Arg Ala Arg Asp Leu Thr 435 440 445 Leu Gin Phe Asp Ala Lys Gly Ser Val Asp Met Glu Tyr Asp Leu Lys 450 455 460 Met Trp Val Trp Gin Ser Pro Thr Pro Val Leu His Thr Val Gly Thr 465 470 475 480 185 173,552/1

Phe Asn Gly Thr Leu 485 Gin Leu Gin His Ser 490 Lys Met Tyr Trp Pro 495 Gly Asn Gin Val Pro val Ser Gin Cys Ser Arg Gin Cys Lys Asp Gly Gin 500 505 510 Val Arg Arg Val Lys Gly Phe His Ser Cys Cys Tyr Asp Cys Val Asp 515 520 525 Cys Lys Ala Gly Ser Tyr Arg Lys His Pro Asp Asp Phe Thr Cys Thr 530 535 540 Pro Cys Gly Lys Asp Gin Trp Ser Pro Glu Lys Ser Thr Thr Cys Leu 545 550 555 560 Pro Arg Arg Pro Lys Phe Leu Glu Trp Gly Glu Pro Ala Val Leu Leu 565 570 575 Leu Leu Leu Leu Leu Ser Leu Ala Leu Gly Leu Val Leu Ala Ala Leu 580 585 - 590 Gly Leu Phe Val His His Arg Asp Ser Pro Leu Val Gin Ala Ser Gly 595 600 605 Gly Pro Leu Ala Cys Phe Gly Leu Val Cys Leu Gly Leu Val Cys Leu 610 615 620 Ser Val Leu Leu Phe Pro Gly Gin Pro Ser Pro Ala Arg Cys Leu Ala 625 630 635 640 Gin Gin Pro Leu Ser His Leu Pro Leu Thr Gly Cys Leu Ser Thr Leu 645 650 655 Phe Leu Gin Ala Ala Glu Ile Phe Val Glu Ser Glu Leu Pro Leu Ser 660 665 670 Trp Ala Asp Arg Leu Ser Gly Cys Leu Arg Gly Pro Trp Ala Trp Leu 675 680 685 Val Val Leu Leu Ala Met Leu Val Glu Val Ala Leu Cys Thr Trp Tyr 690 695 700 Leu Val Ala Phe Pro Pro Glu Val Val Thr Asp Trp His Met Leu Pro 705 710 715 720 Thr Glu Ala Leu Val His Cys Arg Thr Arg Ser Trp Val Ser Phe Gly 725 730 735 Leu Ala His Ala Thr Asn Ala Thr Leu Ala Phe Leu Cys Phe Leu Gly 740 745 750 Thr Phe Leu Val Arg Ser Gin Pro Gly Arg Tyr Asn Arg Ala Arg Gly 755 760 765 Leu Thr Phe Ala Met Leu Ala Tyr Phe Ile Thr Trp Val Ser Phe Val 770 775 780 Pro Leu Leu Ala Asn Val Gin Val Val Leu Arg Pro Ala Val Gin Met 785 790 795 800 Gly Ala Leu Leu Leu Cys Val Leu Gly Ile Leu Ala Ala Phe His Leu 805 810 815 Pro Arg Cys Tyr Leu Leu Met Arg Gin Pro Gly Leu Asn Thr Pro Glu 820 825 830 Phe Phe Leu Gly Gly Gly Pro Gly Asp Ala Gin Gly Gin Asn Asp Gly 835 840 845 Asn Thr Gly Asn Gin Gly Lys His Glu 850 855 &lt;210&gt; 26 &lt;211&gt; 840

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 26

Met Gly Pro Arg Ala Lys Thr Ile Cys Ser Leu Phe Phe Leu Leu Trp 15 10 15 186 173,552/1

Val Leu Ala Glu 20 Pro Ala Glu Asn Ser Asp Phe 25 Tyr Leu Pro 30 Gly Asp Tyr Leu Leu Gly Gly Leu Phe Ser Leu His Ala Asn Met Lys Gly He 35 40 45 Val His Leu Asn Phe Leu Gin Val Pro Met Cys Lys Glu Tyr Glu Val 50 55 60 Lys Val He Gly Tyr Asn Leu Met Gin Ala Met Arg Phe Ala Val Glu 65 70 75 80 Glu He Asn Asn Asp Ser Ser Leu Leu Pro Gly Val Leu Leu Gly Tyr 85 90 95 Glu He Val Asp Val Cys Tyr lie Ser Asn Asn Val Gin Pro Val Leu 100 105 110 Tyr Phe Leu Ala His Glu Asp Asn Leu Leu Pro He Gin Glu Asp Tyr 115 120 125 Ser Asn Tyr He Ser Arg Val Val Ala Val He Gly Pro Asp Asn Ser 130 135 140 Glu Ser Val Met Thr Val Ala Asn Phe Leu Ser Leu Phe Leu Leu Pro 145 150 155 160 Gin He Thr Tyr Ser Ala He Ser Asp Glu Leu Arg Asp Lys Val Arg 165 170 175 Phe Pro Ala Leu Leu Arg Thr Thr Pro Ser Ala Asp His His Val Glu 180 185 190 Ala Met Val Gin Leu Met Leu His Phe Arg Trp Asn Trp He He Val 195 200 205 Leu Val Ser Ser Asp Thr Tyr Gly Arg Asp Asn Gly Gin Leu Leu Gly 210 215 220 Glu Arg Val Ala Arg Arg Asp lie Cys He Ala Phe Gin Glu Thr Leu •225 230 235 240 Pro Thr Leu Gin Pro Asn Gin Asn Met Thr Ser Glu Glu Arg Gin Arg 245 250 255 Leu Val Thr lie Val Asp Lys Leu Gin Gin Ser Thr Ala Arg Val Val 260 265 270 Val Val Phe Ser Pro Asp Leu Thr Leu Tyr His Phe Phe Asn Glu Val 275 280 285 Leu Arg Gin Asn Phe Thr Gly Ala Val Trp He Ala Ser Glu Ser Trp 290 295 300 Ala He Asp Pro Val Leu His Asn Leu Thr Glu Leu Gly His Leu Gly 305 310 315 320 Thr Phe Leu Gly lie Thr He Gin Ser Val Pro He Pro Gly Phe Ser 325 330 335 Glu Phe Arg Glu Trp Gly Pro Gin Ala Gly Pro Pro Pro Leu Ser Arg 340 345 350 Thr Ser Gin Ser Tyr Thr Cys Asn Gin Glu Cys Asp Asn Cys Leu Asn 355 360 365 Ala Thr Leu Ser Phe Asn Thr He Leu Arg Leu Ser Gly Glu Arg Val 370 375 380 Val Tyr Ser Val Tyr Ser Ala Val Tyr Ala Val Ala His Ala Leu His 385 390 395 400 Ser Leu Leu Gly Cys Asp Lys Ser Thr Cys Thr Lys Arg Val Val Tyr 405 410 415 Pro Trp Gin Leu Leu Glu Glu He Trp Lys Val Asn Phe Thr Leu Leu 420 425 430 Asp His Gin He Phe Phe Asp Pro Gin Gly Asp Val Ala Leu His Leu 435 440 445 Glu He Val Gin Trp Gin Trp Asp Arg Ser Gin Asn Pro Phe Gin Ser 450 455 460 Val Ala Ser Tyr Tyr Pro Leu Gin Arg Gin Leu Lys Asn He Gin Asp 465 470 475 480 lie Ser Trp His Thr Val Asn Asn Thr He Pro Met Ser Met Cys Ser 485 490 495 187 173,552/1

Lys Arg Cys Gin 500 Ser Gly Gin Lys Lys 505 Lys Pro Val Gly Ile 510 His Val Cys Cys Phe Glu Cys Ile Asp Cys Leu Pro Gly Thr Phe Leu Asn His 515 520 525 Thr Glu Asp Glu Tyr Glu Cys Gin Ala Cys Pro Asn Asn Glu Trp Ser 530 535 540 Tyr Gin Ser Glu Thr Ser Cys Phe Lys Arg Gin Leu Val Phe Leu Glu 545 550 555 560 Leu Arg Glu His Thr Ser Trp Val Leu Leu Ala Ala Asn Thr Leu Leu 565 570 575 Leu Leu Leu Leu Leu Gly Thr Ala Gly Leu Phe Ala Trp His Leu Asp 580 585 590 Thr Pro Val Val Arg Ser Ala Gly Gly Arg Leu Cys Phe Leu Met Leu 595 600 605 Gly Ser Leu Ala Ala Gly Ser Gly Ser Leu Tyr Gly Phe Phe Gly Glu 610 615 620 Pro Thr Arg Pro Ala Cys Leu Leu Arg Gin Ala Leu Phe Ala Leu Gly 625 630 635 640 Phe Thr Ile Phe Leu Ser Cys Leu Thr Val Arg Ser Phe Gin Leu Ile 645 650 655 Ile Ile Phe Lys Phe Ser Thr Lys Val Pro Thr Phe Tyr His Ala Trp 660 665 670 Val Gin Asn His Gly Ala Gly Leu Phe Val Met Ile Ser Ser Ala Ala 675 680 685 Gin Leu Leu Ile Cys Leu Thr Trp Leu Val Val Trp Thr Pro Leu Pro 690 695 700 Ala Arg Glu Tyr Gin Arg Phe Pro His Leu Val Met Leu Glu Cys Thr 705 710 715 720 Glu Thr Asn Ser Leu Gly Phe Ile Leu Ala Phe Leu Tyr Asn Gly Leu 725 730 735 Leu Ser Ile Ser Ala Phe Ala Cys Ser Tyr Leu Gly Lys Asp Leu Pro 740 745 750 Glu Asn Tyr Asn Glu Ala Lys Cys Val Thr Phe Ser Leu Leu Phe Asn 755 760 765 Phe Val Ser Trp Ile Ala Phe Phe Thr Thr Ala Ser Val Tyr Asp Gly 770 775 780 Lys Tyr Leu Pro Ala Ala Asn Met Met Ala Gly Leu Ser Ser Leu Ser 785 790 795 800 Ser Gly Phe Gly Gly Tyr Phe Leu Pro Lys Cys Tyr Val Ile Leu Cys 805 810 815 Arg Pro Asp Leu Asn Ser Thr Glu His Phe Gin Ala Ser Ile Gin Asp 820 825 830 Tyr Thr Arg Arg Cys Gly Ser Thr 835 840 &lt;210&gt; 27 &lt;211&gt; 840

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 27

Met Leu Leu Cys Thr Ala Arg Leu Val Gly Leu Gin Leu Leu Ile Ser 1 5 10 15 Cys Cys Trp Ala Phe Ala Cys His Ser Thr Glu Ser Ser Pro Asp Phe 20 25 30 Thr Leu Pro Gly Asp Tyr Leu Leu Ala Gly Leu Phe Pro Leu His Ser 35 40 45 188 173,552/1

Gly Cys 50 Leu Gin Val Arg His 55 Arg Pro Glu Val Thr 60 Leu Cys Asp Arg Ser Cys Ser Phe Asn Glu His Gly Tyr His Leu Phe Gin Ala Met Arg 65 70 75 80 Leu Gly Val Glu Glu He Asn Asn Ser Thr Ala Leu Leu Pro Asn He 85 90 95 Thr Leu Gly Tyr Gin Leu Tyr Asp Val Cys Ser Asp Ser Ala Asn Val 100 105 110 Tyr Ala Thr Leu Arg Val Leu Ser Leu Pro Gly Gin His His He Glu 115 120 125 Leu Gin Gly Asp Leu Leu His Tyr Ser Pro Thr Val Leu Ala Val He 130 135 140 Gly Pro Asp Ser Thr Asn Arg Ala Ala Thr Thr Ala Ala Leu Leu Ser 145 150 155 160 Pro Phe Leu Val Pro Met He Ser Tyr Ala Ala Ser Ser Glu Thr Leu 165 170 175 Ser Val Lys Arg Gin Tyr Pro Ser Phe Leu Arg Thr He Pro Asn Asp 180 185 190 Lys Tyr Gin Val Glu Thr Met Val Leu Leu Leu Gin Lys Phe Gly Trp 195 200 205 Thr Trp lie Ser Leu Val Gly Ser Ser Asp Asp Tyr Gly Gin Leu Gly 210 215 220 Val Gin Ala Leu Glu Asn Gin Ala Thr Gly Gin Gly He Cys He Ala 225 230 235 240 Phe Lys Asp He Met Pro Phe Ser Ala Gin Val Gly Asp Glu Arg Met 245 250 255 Gin Cys Leu Met Arg His Leu Ala Gin Ala Gly Ala Thr Val Val Val 260 265 270 Val Phe Ser Ser Arg Gin Leu Ala Arg Val Phe Phe Glu Ser Val Val 275 280 285 Leu Thr Asn Leu Thr Gly Lys Val Trp Val Ala Ser Glu Ala Trp Ala 290 295 300 Leu Ser Arg His lie Thr Gly Val Pro Gly He Gin Arg He Gly Met 305 310 315 320 Val Leu Gly Val Ala He Gin Lys Arg Ala Val Pro Gly Leu Lys Ala 325 330 335 Phe Glu Glu Ala Tyr Ala Arg Ala Asp Lys Lys Ala Pro Arg Pro Cys 340 345 350 His Lys Gly Ser Trp Cys Ser Ser Asn Gin Leu Cys Arg Glu Cys Gin 355 360 365 Ala Phe Met Ala His Thr Met Pro Lys Leu Lys Ala Phe Ser Met Ser 370 375 380 Ser Ala Tyr Asn Ala Tyr Arg Ala Val Tyr Ala Val Ala His Gly Leu 385 390 395 400 His Gin Leu Leu Gly Cys Ala Ser Gly Ala Cys Ser Arg Gly Arg Val 405 410 415 Tyr Pro Trp Gin Leu Leu Glu Gin He His Lys Val His Phe Leu Leu 420 425 430 His Lys Asp Thr Val Ala Phe Asn Asp Asn Arg Asp Pro Leu Ser Ser 435 440 445 Tyr Asn He He Ala Trp Asp Trp Asn Gly Pro Lys Trp Thr Phe Thr 450 455 460 Val Leu Gly Ser Ser Thr Trp Ser Pro Val Gin Leu Asn He Asn Glu 465 470 475 480 Thr Lys He Gin Trp His Gly Lys Asp Asn Gin Val Pro Lys Ser Val 485 490 495 Cys Ser Ser Asp Cys Leu Glu Gly His Gin Arg Val Val Thr Gly Phe 500 505 510 His His Cys Cys Phe Glu Cys Val Pro Cys Gly Ala Gly Thr Phe Leu 515 520 525 189 173,552/1

Asn Lys 530 Ser Asp Leu Tyr Arg 535 Cys Gin Pro Cys Gly 540 Lys Glu Glu Trp Ala Pro Glu Gly Ser Gin Thr Cys Phe Pro Arg Thr Val Val Phe Leu 545 550 555 560 Glu Trp His Glu Ala Pro Thr Ile Ala Val Ala Leu Leu Ala Ala Leu 565 570 575 Gly Phe Leu Ser Thr Leu Ala Ile Leu Val Ile Phe Trp Arg His Phe 580 585 590 Gin Thr Pro Ile Val Arg Ser Ala Gly Gly Pro Met Cys Phe Leu Met 595 600 605 Leu Thr Leu Leu Leu Val Ala Tyr Met Val Val Pro Val Tyr Val Gly 610 615 620 Pro Pro Lys Val Ser Thr Cys Leu Cys Arg Gin Ala Leu Phe Pro Leu 625 630 635 640 Cys Phe Thr Ile Cys lie Ser Cys Ile Ala Val Arg Ser Phe Gin Ile 645 650 655 Val Cys Ala Phe Lys Met Ala Ser Arg Phe Pro Arg Ala Tyr Ser Tyr 660 665 670 Trp Val Arg Tyr Gin Gly Pro Tyr Val Ser Met Ala Phe Ile Thr Val 675 680 685 Leu Lys Met Val Ile Val Val Ile Gly Met Leu Ala Thr Gly Leu Ser 690 695 700 Pro Thr Thr Arg Thr Asp Pro Asp Asp Pro Lys Ile Thr lie Val Ser 705 710 715 720 Cys Asn Pro Asn Tyr Arg Asn Ser Leu Leu Phe Asn Thr Ser Leu Asp 725 730 735 Leu Leu Leu Ser Val Val Gly Phe Ser Phe Ala Tyr Met Gly Lys Glu 740 745 750 Leu Pro Thr Asn Tyr Asn Glu Ala Lys Phe Ile Thr Leu Ser Met Thr 755 760 765 Phe Tyr Phe Thr Ser Ser Val Ser Leu Cys Thr Phe Met Ser Ala Tyr 770 775 780 Ser Gly Val Leu Val Thr Ile Val Asp Leu Leu Val Thr Val Leu Asn 785 790 795 800 Leu Leu Ala Ile Ser Leu Gly Tyr Phe Gly Pro Lys Cys Tyr Met Ile 805 810 815 Leu Phe Tyr Pro Glu Arg Asn Thr Pro Ala Tyr Phe Asn Ser Met lie 820 825 830 Gin Gly Tyr Thr Met Arg Arg Asp 835 840 &lt;210&gt; 28 &lt;211&gt; 1123

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 28 Leu Gin Val Arg His Arg Pro Glu Val Thr Leu Cys Asp Arg Ser Cys 1 Ser 5 10 15 Phe Asn Glu His Gly Tyr His Leu Phe Gin Ala Met Arg Leu Gly Val 20 25 30 Glu Glu Ile Asn Asn Ser Thr Ala Leu Leu Pro Asn Ile Thr Leu Gly 35 40 45 Tyr Gin Leu Tyr Asp Val Cys Ser Asp Ser Ala Asn Val Tyr Ala Thr 50 55 60 Leu Arg Val Leu Ser Leu Pro Gly Gin His His Ile Glu Leu Gin 65 70 75 80 190 173,552/1

Gly Asp Leu Leu Asn 100 Met His 85 Arg He Tyr Ala Ser Ser Ala Tyr Pro Thr Ala Thr Val 90 Thr Ala 105 Leu Ala Val He Ser 110 Leu Gly 95 Pro Ser Pro Phe Val Asp Leu Ser Val Thr Pro Ala Ser Leu Glu Leu Thr Ala Ser 115 120 125 Lys Arg Gin Tyr Pro Ser Phe Leu Arg Thr He Pro Asn Asp Lys Tyr 130 135 140 Gin Val Glu Thr Met Val Leu Leu Leu Gin Lys Phe Gly Trp Thr Trp 145 150 155 160 Xie Ser Leu Val Gly Ser Ser Asp Asp Tyr Gly Gin Leu Gly Val Gin 165 170 175 Ala Leu Glu Asn Gin Ala Thr Gly Gin Gly lie Cys He Ala Phe Lys 180 185 190 Asp lie Met Pro Phe Ser Ala Gin Val Gly Asp Glu Arg Met Gin Cys 195 200 205 Leu Met Arg His Leu Ala Gin Ala Gly Ala Thr Val Val Val Val Phe 210 215 220 Ser Ser Arg Gin Leu Ala Arg Val Phe Phe Glu Ser Val Val Leu Thr 225 230 235 240 Asn Leu Thr Gly Lys Val Trp Val Ala Ser Glu Ala Trp Ala Leu Ser 245 250 255 Arg His He Thr Gly Val Pro Gly He Gin Arg He Gly Met Val Leu 260 265 270 Gly Val Ala He Gin Lys Arg Ala Val Pro Gly Leu Lys Ala Phe Glu 275 280 285 Glu Ala Tyr Ala Arg Ala Asp Lys Lys Ala Pro Arg Pro Cys His Lys 290 295 300 Gly Ser Trp Cys Ser Ser Asn Gin Leu Cys Arg Glu Cys Gin Ala Phe 305 310 315 320 Met Ala His Thr Met Pro Lys Leu Lys Ala Phe Ser Met Ser Ser Ala 325 330 335 Tyr Asn Ala Tyr Arg Ala Val Tyr Ala Val Ala His Gly Leu His Gin 340 345 350 Leu Leu Gly Cys Ala Ser Gly Ala Cys Ser Arg Gly Arg Val Tyr Pro 355 360 365 Trp Gin Leu Leu Glu Gin He His Lys Val His Phe Leu Leu His Lys 370 375 380 Asp Thr Val Ala Phe Asn Asp Asn Arg Asp Pro Leu Ser Ser Tyr Asn 385 390 395 400 Xie He Ala Trp Asp Trp Asn Gly Pro Lys Trp Thr Phe Thr Val Leu 405 410 415 Gly Ser Ser Thr Trp Ser Pro Val Gin Leu Asn He Asn Glu Thr Lys 420 425 430 lie Gin Trp His Gly Lys Asp Asn Gin Val Pro Lys Ser Val Cys Ser 435 440 445 Ser Asp Cys Leu Glu Gly His Gin Arg Val Val Thr Gly Phe His His 450 455 460 Cys Cys Phe Glu Cys Val Pro Cys Gly Ala Gly Thr Phe Leu Asn Lys 465 470 475 480 Ser Asp Leu Tyr Arg Cys Gin Pro Cys Gly Lys Glu Glu Trp Ala Pro 485 490 495 Glu Gly Ser Gin Thr Cys Phe Pro Arg Thr Val Val Phe Leu Glu Trp 500 505 510 Ser Asp lie Glu Ser He He Ala lie Ala Phe Ser Cys Leu Gly He 515 520 525 Leu Val Thr Leu Phe Val Thr Leu He Phe Val Leu Tyr Arg Asp Thr 530 535 540 Pro Val Val Lys Ser Ser Ser Arg Glu Leu Cys Tyr lie He Leu Ala 545 550 555 560 191 173,552/1

Gly He Phe Leu Gly 565 Tyr Val Cys Pro Phe 570 Thr Leu He Ala Lys 575 Pro Thr Thr Thr Ser Cys Tyr Leu Gin Arg Leu Leu Val Gly Leu Ser Ser 580 585 590 Ala Met Cys Tyr Ser Ala Leu Val Thr Lys Thr Asn Arg He Ala Arg 595 600 605 lie Leu Ala Gly Ser Lys Lys Lys He Cys Thr Arg Lys Pro Arg Phe 610 615 620 Met Ser Ala Trp Ala Gin Val He lie Ala Ser He Leu He Ser Val 625 630 635 640 Gin Leu Thr Leu Val Val Thr Leu He He Met Glu Pro Pro Met Pro 645 650 655 He Leu Ser Tyr Pro Ser He Lys Glu Val Tyr Leu Xie Cys Asn Thr 660 665 670 Ser Asn Leu Gly Val Val Ala Pro Val Gly Tyr Asn Gly Leu Leu He 675 680 685 Met Ser Cys Thr Tyr Tyr Ala Phe Lys Thr Arg Asn Val Pro Ala Asn 690 695 700 Phe Asn Glu Ala Lys Tyr He Ala Phe Thr Met Tyr Thr Thr Cys He 705 710 715 720 He Trp Leu Ala Phe Val Pro lie Tyr Phe Gly Ser Asn Tyr Lys He 725 730 735 lie Thr Thr Cys Phe Ala Val Ser Leu Ser Val Thr Val Ala Leu Gly 740 745 750 Cys Met Phe Thr Pro Lys Met Tyr He He He Ala Lys Pro Glu Arg 755 760 765 Asn Val Arg Ser Ala Phe Thr Thr Ser Asp Val Val Arg Met His Val 770 775 780 Gly Asp Gly Lys Leu Pro Cys Arg Ser Asn Thr Phe Leu Asn lie Phe 785 790 795 800 Arg Arg Lys Lys Pro Gly Ala Gly Asn Ala Asn Ser Asn Gly Lys Ser 805 810 815 Val Ser Trp Ser Glu Pro Gly Gly Arg Gin Ala Pro Lys Gly Gin His 820 825 830 Val Trp Gin Arg Leu Ser Val His Val Lys Thr Asn Glu Thr Ala Cys 835 840 845 Asn Gin Thr Ala Val He Lys Pro Leu Thr Lys Ser Tyr Gin Gly Ser 850 855 860 Gly Lys Ser Leu Thr Phe Ser Asp Ala Ser Thr Lys Thr Leu Tyr Asn 865 870 875 880 Val Glu Glu Glu Asp Asn Thr Pro Ser Ala His Phe Ser Pro Pro Ser 885 890 895 Ser Pro Ser Met Val Val His Arg Arg Gly Pro Pro Val Ala Thr Thr 900 905 910 Pro Pro Leu Pro Pro His Leu Thr Ala Glu Glu Thr Pro Leu Phe Leu 915 920 925 Ala Asp Ser Val He Pro Lys Gly Leu Pro Pro Pro Leu Pro Gin Gin 930 935 940 Gin Pro Gin Gin Pro Pro Pro Gin Gin Pro Pro Gin Gin Pro Lys Ser 945 950 955 960 Leu Met Asp Gin Leu Gin Gly Val Val Thr Asn Phe Gly Ser Gly He 965 970 975 Pro Asp Phe His Ala Val Leu Ala Gly Pro Gly Thr Pro Gly Asn Ser 980 985 990 Leu Arg Ser Leu Tyr Pro Pro Pro Pro Pro Pro Gin His Leu Gin Met 995 1000 1005 Leu Pro Leu His Leu Ser Thr Phe Gin Glu Glu Ser He Ser Pro Pro 1010 1015 1020 Gly Glu Asp He Asp Asp Asp Ser Glu Arg Phe Lys Leu Leu Gin Glu 1025 1030 1035 1040 192 173,552/1

Phe Val Tyr Glu Arg Glu 1045 Gly Asn Thr Glu Glu Asp 1050 Glu Leu Glu Glu 1055 Glu Glu Asp Leu Pro Thr Ala Ser Lys Leu Thr Pro Glu Asp Ser Pro 1060 1065 1070 Ala Leu Thr Pro Pro Ser Pro Phe Arg Asp Ser Val Ala Ser Gly Ser 1075 1080 1085 Ser Val Pro Ser Ser Pro Val Ser Glu Ser Val Leu Cys Thr Pro Pro 1090 1095 1100 Asn Val Thr Tyr Ala Ser Val lie Leu Arg Asp Tyr Lys Gin Ser Ser 1105 1110 1115 1120 Ser Thr Leu &lt;210&gt; 29 &lt;211&gt; 1172 &lt;212&gt; PRT &lt;213&gt; Artificial . Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 29 Met Gly Pro Arg Ala Lys Thr He Cys Ser Leu Phe Phe Leu Leu Trp 1 5 10 15 Val Leu Ala Glu Pro Ala Glu Asn Ser Asp Phe Tyr Leu Pro Gly Asp 20 25 30 Tyr Leu Leu Gly Gly Leu Phe Ser Leu His Ala Asn Met Lys Gly He 35 40 45 Val His Leu Asn Phe Leu Gin Val Pro Met Cys Lys Glu Tyr Glu Val 50 55 60 Lys Val He Gly Tyr Asn Leu Met Gin Ala Met Arg Phe Ala Val Glu 65 70 75 80 Glu He Asn Asn Asp Ser Ser Leu Leu Pro Gly Val Leu Leu Gly Tyr 85 90 95 Glu He Val Asp Val Cys Tyr He Ser Asn Asn Val Gin Pro Val Leu 100 105 110 Tyr Phe Leu Ala His Glu Asp Asn Leu Leu Pro He Gin Glu Asp Tyr 115 120 125 Ser Asn Tyr He Ser Arg Val Val Ala Val He Gly Pro Asp Asn Ser 130 135 140 Glu Ser Val Met Thr Val Ala Asn Phe Leu Ser Leu Phe Leu Leu Pro 145 150 155 160 Gin He Thr Tyr Ser Ala He Ser Asp Glu Leu Arg Asp Lys Val Arg 165 170 175 Phe Pro Ala Leu Leu Arg Thr Thr Pro Ser Ala Asp His His Val Glu 180 185 190 Ala Met Val Gin Leu Met Leu His Phe Arg Trp Asn Trp He He Val 195 200 205 Leu Val Ser Ser Asp Thr Tyr Gly Arg Asp Asn Gly Gin Leu Leu Gly 210 215 220 Glu Arg Val Ala Arg Arg Asp lie Cys He Ala Phe Gin Glu Thr Leu 225 230 235 240 Pro Thr Leu Gin Pro Asn Gin Asn Met Thr Ser Glu Glu Arg Gin Arg 245 250 255 Leu Val Thr He Val Asp Lys Leu Gin Gin Ser Thr Ala Arg Val Val 260 265 270 Val Val Phe Ser Pro Asp Leu Thr Leu Tyr His Phe Phe Asn Glu Val 275 280 285 Leu Arg Gin Asn Phe Thr Gly Ala Val Trp He Ala Ser Glu Ser Trp 290 295 300 193 173,552/1

Ala 305 Ile Asp Pro Val Leu 310 His Asn Leu Thr Glu 315 Leu Gly His Leu Gly 320 Thr Phe Leu Gly Ile Thr Ile Gin Ser Val Pro Ile Pro Gly Phe Ser 325 330 335 Glu Phe Arg Glu Trp Gly Pro Gin Ala Gly Pro Pro Pro Leu Ser Arg 340 345 350 Thr Ser Gin Ser Tyr Thr Cys Asn Gin Glu Cys Asp Asn Cys Leu Asn 355 360 365 Ala Thr Leu Ser Phe Asn Thr Ile Leu Arg Leu Ser Gly Glu Arg Val 37.0 375 380 Val Tyr Ser Val Tyr Ser Ala Val Tyr Ala Val Ala His Ala Leu His 385 390 395 400 Ser Leu Leu Gly Cys Asp Lys Ser Thr Cys Thr Lys Arg Val Val Tyr 405 410 415 Pro Trp Gin Leu Leu Glu Glu Ile Trp Lys Val Asn Phe Thr Leu Leu 420 425 430 Asp His Gin Ile Phe Phe Asp Pro Gin Gly Asp Val Ala Leu His Leu 435 440 445 Glu Ile Val Gin Trp Gin Trp Asp Arg Ser Gin Asn Pro Phe Gin Ser 450 455 460 Val Ala Ser Tyr Tyr Pro Leu Gin Arg Gin Leu Lys Asn Ile Gin Asp 465 470 475 480 Ile Ser Trp His Thr Val Asn Asn Thr Ile Pro Met Ser Met Cys Ser 485 490 495 Lys Arg Cys Gin Ser Gly Gin Lys Lys Lys Pro Val Gly Ile His Val 500 505 510 Cys Cys Phe Glu Cys Ile Asp Cys Leu Pro Gly Thr Phe Leu Asn His 515 520 525 Thr Glu Asp Glu Tyr Glu Cys Gin Ala Cys Pro Asn Asn Glu Trp Ser 530 535 540 Tyr Gin Ser Glu Thr Ser Cys Phe Lyh Arg Gin Leu Val Phe Leu Glu 545 550 555 560 Trp Ser Asp Ile Glu Ser Ile Ile Ala Ile Ala Phe Ser Cys Leu Gly 565 570 575 Ile Leu Val Thr Leu Phe Val Thr Leu Ile Phe Val Leu Tyr Arg Asp 580 585 590 Thr Pro Val Val Lys Ser Ser Ser Arg Glu Leu Cys Tyr lie Ile Leu 595 600 605 Ala Gly Ile Phe Leu Gly Tyr Val Cys Pro Phe Thr Leu Ile Ala Lys 610 615 620 Pro Thr Thr Thr Ser Cys Tyr Leu Gin Arg Leu Leu Val Gly Leu Ser 625 630 635 640 Ser Ala Met Cys Tyr Ser Ala Leu Val Thr Lys Thr Asn Arg Ile Ala 645 650 655 Arg Ile Leu Ala Gly Ser Lys Lys Lys Ile Cys Thr Arg Lys Pro Arg 660 665 670 Phe Met Ser Ala Trp Ala Gin Val Ile Ile Ala Ser Ile Leu Ile Ser 675 680 685 Val Gin Leu Thr Leu Val Val Thr Leu Ile Ile Met Glu Pro Pro Met 690 695 700 Pro Ile Leu Ser Tyr Pro Ser lie Lys Glu Val Tyr Leu Ile Cys Asn 705 710 715 720 Thr Ser Asn Leu Gly Val Val Ala Pro Val Gly Tyr Asn Gly Leu Leu 725 730 735 Ile Met Ser Cys Thr Tyr Tyr Ala Phe Lys Thr Arg Asn Val Pro Ala 740 745 750 Asn Phe Asn Glu Ala Lys Tyr Ile Ala Phe Thr Met Tyr Thr Thr Cys 755 760 765 Ile Ile Trp Leu Ala Phe Val Pro Ile Tyr Phe Gly Ser Asn Tyr Lys 770 775 780 194 173,552/1 lie He Thr Thr Cys Phe Ala Val Ser Leu Ser Val Thr Val Ala Leu 785 790 795 800 Gly Cys Met Phe Thr Pro Lys Met Tyr lie He lie Ala Lys Pro Glu 805 810 815 Arg Asn Val Arg Ser Ala Phe Thr Thr Ser Asp Val Val Arg Met His 820 825 830 Val Gly Asp Gly Lys Leu Pro Cys Arg Ser Asn Thr Phe Leu Asn lie 835 840 845 Phe Arg Arg Lys Lys Pro Gly Ala Gly Asn Ala Asn Ser Asn Gly Lys 850 855 860 Ser Val Ser Trp Ser Glu Pro Gly Gly Arg Gin Ala Pro Lys Gly Gin 865 870 875 880 His Val Trp Gin Arg Leu Ser Val His Val Lys Thr Asn Glu Thr Ala 885 890 895 Cys Asn Gin Thr Ala Val lie Lys Pro Leu Thr Lys Ser Tyr Gin Gly 900 905 910 Ser Gly Lys Ser Leu Thr Phe Ser Asp Ala Ser Thr Lys Thr Leu Tyr 915 920 925 Asn Val Glu Glu Glu Asp Asn Thr Pro Ser Ala His Phe Ser Pro Pro 930 935 940 Ser Ser Pro Ser Met Val Val His Arg Arg Gly Pro Pro Val Ala Thr 945 950 955 960 Thr Pro Pro Leu Pro Pro His Leu Thr Ala Glu Glu Thr Pro Leu Phe 965 970 975 Leu Ala Asp Ser Val lie Pro Lys Gly Leu Pro Pro Pro Leu Pro Gin 980 985 990 Gin Gin Pro Gin Gin Pro Pro Pro Gin Gin Pro Pro Gin Gin Pro Lys 995 1000 1005 Ser Leu Met Asp Gin Leu Gin Gly Val Val Thr Asn Phe Gly Ser Gly 1010 1015 1020 He Pro Asp Phe His Ala Val Leu Ala Gly Pro Gly Thr Pro Gly Asn 1025 1030 1035 1040 Ser Leu Arg Ser Leu Tyr Pro Pro Pro Pro Pro Pro Gin His Leu Gin 1045 1050 1055 Met Leu Pro Leu His Leu Ser Thr Phe Gin Glu Glu Ser lie Ser Pro 1060 1065 1070 Pro Gly Glu Asp lie Asp Asp Asp Ser Glu Arg Phe Lys Leu Leu Gin 1075 1080 1085 Glu Phe Val Tyr Glu Arg Glu Gly Asn Thr Glu Glu Asp Glu Leu Glu 1090 1095 1100 Glu Glu Glu Asp Leu Pro Thr Ala Ser Lys Leu Thr Pro Glu Asp Ser 1105 1110 1115 1120 Pro Ala Leu Thr Pro Pro Ser Pro Phe Arg Asp Ser Val Ala Ser Gly 1125 1130 1135 Ser Ser Val Pro Ser Ser Pro Val Ser Glu Ser Val Leu Cys Thr Pro 1140 1145 1150 Pro Asn Val Thr Tyr Ala Ser Val lie Leu Arg Asp Tyr Lys Gin Ser 1155 1160 1165 Ser Ser Thr Leu 1170 &lt;210&gt; 30 &lt;211&gt; 1175

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 30 195 173,552/1

Met Leu Gly Pro Ala Val Leu Gly Leu Ser Leu Trp Ala Leu Leu His 1 5 10 15 Pro Gly Thr Gly Ala Pro Leu Cys Leu Ser Gin Gin Leu Arg Met Lys 20 25 30 Gly Asp Tyr Val Leu Gly Gly Leu Phe Pro Leu Gly Glu Ala Glu Glu 35 40 45 Ala Gly Leu Arg Ser Arg Thr Arg Pro Ser Ser Pro Val Cys Thr Arg 50 55 60 Phe Ser Ser Asn Gly Leu Leu Trp Ala Leu Ala Met Lys Met Ala Val 65 70 75 80 Glu Glu Ile Asn Asn Lys Ser Asp Leu Leu Pro Gly Leu Arg Leu Gly 85 90 95 Tyr Asp Leu Phe Asp Thr Cys Ser Glu Pro Val Val Ala Met Lys Pro 100 105 110 Ser Leu Met Phe Leu Ala Lys Ala Gly Ser Arg Asp Ile Ala Ala Tyr 115 120 125 Cys Asn Tyr Thr Gin Tyr Gin Pro Arg Val Leu Ala Val Ile Gly Pro 130 135 140 His Ser Ser Glu Leu Ala Met Val Thr Gly Lys Phe Phe Ser Phe Phe 145 150 155 160 Leu Met Pro Gin Val Ser Tyr Gly Ala Ser Met Glu Leu Leu Ser Ala 165 170 175 Arg Glu Thr Phe Pro Ser Phe Phe Arg Thr Val Pro Ser Asp Arg Val 180 185 190 Gin Leu Thr Ala Ala Ala Glu Leu Leu Gin Glu Phe Gly Trp Asn Trp 195 200 205 Val Ala Ala Leu Gly Ser Asp Asp Glu Tyr Gly Arg Gin Gly Leu Ser 210 215 220 Ile Phe Ser Ala Leu Ala Ala Ala Arg Gly Ile Cys Ile Ala His Glu 225 230 235 240 Gly Leu Val Pro Leu Pro Arg Ala Asp Asp Ser Arg Leu Gly Lys Val 245 250 255 Gin Asp Val Leu His Gin Val Asn Gin Ser Ser Val Gin Val Val Leu 260 265 270 Leu Phe Ala Ser Val His Ala Ala His Ala Leu Phe Asn Tyr Ser Ile 275 280 285 Ser Ser Arg Leu Ser Pro Lys Val Trp Val Ala Ser Glu Ala Trp Leu 290 295 300 Thr Ser Asp Leu Val Met Gly Leu Pro Gly Met Ala Gin Met Gly Thr 305 310 315 320 Val Leu Gly Phe Leu Gin Arg Gly Ala Gin Leu His Glu Phe Pro Gin 325 330 335 Tyr Val Lys Thr His Leu Ala Leu Ala Thr Asp Pro Ala Phe Cys Ser 340 345 350 Ala Leu Gly Glu Arg Glu Gin Gly Leu Glu Glu Asp Val Val Gly Gin 355 360 365 Arg Cys Pro Gin Cys Asp Cys lie Thr Leu Gin Asn Val Ser Ala Gly 370 375 380 Leu Asn His His Gin Thr Phe Ser Val Tyr Ala Ala Val Tyr Ser Val 385 390 395 400 Ala Gin Ala Leu His Asn Thr Leu Gin Cys Asn Ala Ser Gly Cys Pro 405 410 415 Ala Gin Asp Pro Val Lys Pro Trp Gin Leu Leu Glu Asn Met Tyr Asn 420 425 430 Leu Thr Phe His Val Gly Gly Leu Pro Leu Arg Phe Asp Ser Ser Gly 435 440 445 Asn Val Asp Met Glu Tyr Asp Leu Lys Leu Trp Val Trp Gin Gly Ser 450 455 460 Val Pro Arg Leu His Asp Val Gly Arg Phe Asn Gly Ser Leu Arg Thr 465 470 475 480 196 173,552/1

Glu Arg Leu Lys He 485 Arg Trp His Thr Ser Asp Asn Gin 490 Lys Pro 495 Val Ser Arg Cys Ser Arg Gin Cys Gin Glu Gly Gin Val Arg Arg Val Lys 500 505 510 Gly Phe His Ser Cys Cys Tyr Asp Cys Val Asp Cys Glu Ala Gly Ser 515 520 525 Tyr Arg Gin Asn Pro Asp Asp He Ala Cys Thr Phe Cys Gly Gin Asp 530 535 540 Glu Trp Ser Pro Glu Arg Ser Thr Arg Cys Phe Arg Arg Arg Ser Arg 545 550 555 560 Phe Leu Glu Trp Ser Asp He Glu Ser He lie Ala lie Ala Phe Ser 565 570 575 Cys Leu Gly He Leu Val Thr Leu Phe Val Thr Leu He Phe Val Leu 580 585 590 Tyr Arg Asp Thr Pro Val Val Lys Ser Ser Ser Arg Glu Leu Cys Tyr 595 600 605 lie He Leu Ala dy lie Phe Leu Gly Tyr Val Cys Pro Phe Thr Leu 610 615 620 He Ala Lys Pro Thr Thr Thr Ser Cys Tyr Leu Gin Arg Leu Leu Val 625 630 635 640 Gly Leu Ser Ser Ala Met Cys Tyr Ser Ala Leu Val Thr Lys Thr Asn 645 650 655 Arg He Ala Arg He Leu Ala Gly Ser Lys Lys Lys He Cys Thr Arg 660 665 670 Lys Pro Arg Phe Met Ser Ala Trp Ala Gin Val He He Ala Ser He 675 680 685 Leu He Ser Val Gin Leu Thr Leu Val Val Thr Leu lie He Met Glu 690 695 700 Pro Pro Met Pro He Leu Ser Tyr Pro Ser He Lys Glu Val Tyr Leu 705 710 715 720 He Cys Asn Thr Ser Asn Leu Gly Val Val Ala Pro Val Gly Tyr Asn 725 730 735 Gly Leu Leu lie Met Ser Cys Thr Tyr Tyr Ala Phe Lys Thr Arg Asn 740 745 750 Val Pro Ala Asn Phe Asn Glu Ala Lys Tyr He Ala Phe Thr Met Tyr 755 760 765 Thr Thr Cys He He Trp Leu Ala Phe Val Pro lie Tyr Phe Gly Ser 770 775 780 Asn Tyr Lys He lie Thr Thr Cys Phe Ala Val Ser Leu Ser Val Thr 785 790 795 800 Val Ala Leu Gly Cys Met Phe Thr Pro Lys Met Tyr He He He Ala 805 810 815 Lys Pro Glu Arg Asn Val Arg Ser Ala Phe Thr Thr Ser Asp Val Val 820 825 830 Arg Met His Val Gly Asp Gly Lys Leu Pro Cys Arg Ser Asn Thr Phe 835 840 845 Leu Asn lie Phe Arg Arg Lys Lys Pro Gly Ala Gly Asn Ala Asn Ser 850 855 860 Asn Gly Lys Ser Val Ser Trp Ser Glu Pro Gly Gly Arg Gin Ala Pro 865 870 875 880 Lys Gly Gin His Val Trp Gin Arg Leu Ser Val His Val Lys Thr Asn 885 890 895 Glu Thr Ala Cys Asn Gin Thr Ala Val lie Lys Pro Leu Thr Lys Ser 900 905 910 Tyr Gin Gly Ser Gly Lys Ser Leu Thr Phe Ser Asp Ala Ser Thr Lys 915 920 925 Thr Leu Tyr Asn Val Glu Glu Glu Asp Asn Thr Pro Ser Ala His Phe 930 935 940 Ser Pro Pro Ser Ser Pro Ser Met Val Val His Arg Arg Gly Pro Pro 945 950 955 960 197 173,552/1

Val Ala Thr Thr Pro 965 Pro Leu Pro Pro His 970 Leu Thr Ala Glu Glu 975 Thr Pro Leu Phe Leu Ala Asp Ser Val Ile Pro Lys Gly Leu Pro Pro Pro 980 985 990 Leu Pro Gin Gin Gin Pro Gin Gin Pro Pro Pro Gin Gin Pro Pro Gin 995 1000 1005 Gin Pro Lys Ser Leu Met Asp Gin Leu Gin Gly Val Val Thr Asn Phe 1010 1015 1020 Gly Ser Gly Ile Pro Asp Phe His Ala Val Leu Ala Gly Pro Gly Thr 1025 1030 1035 1040 Pro Gly Asn Ser Leu Arg Ser Leu Tyr Pro Pro Pro Pro Pro Pro Gin 1045 1050 1055 His Leu Gin Met Leu Pro Leu His Leu Ser Thr Phe Gin Glu Glu Ser 1060 1065 1070 Xie Ser Pro Pro Gly Glu Asp Ile Asp Asp Asp Ser Glu Arg Phe Lys 1075 1080 1085 Leu Leu Gin Glu Phe Val Tyr Glu Arg Glu Gly Asn Thr Glu Glu Asp 1090 1095 1100 Glu Leu Glu Glu Glu Glu Asp Leu Pro Thr Ala Ser Lys Leu Thr Pro 1105 1110 1115 1120 Glu Asp Ser Pro Ala Leu Thr Pro Pro Ser Pro Phe Arg Asp Ser Val 1125 1130 1135 Ala Ser Gly Ser Ser Val Pro Ser Ser Pro Val Ser Glu Ser Val Leu 1140 1145 1150 Cys Thr Pro Pro Asn Val Thr Tyr Ala Ser Val Ile Leu Arg Asp Tyr 1155 1160 1165 Lys Gin Ser Ser Ser Thr Leu 1170 1175 &lt;210&gt; 31 &lt;211&gt; 867

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 31

Met 1 Val Arg Leu Leu 5 Leu Ile Phe Phe Pro 10 Met Ile Phe Leu Glu 15 Met Ser lie Leu Pro Arg Met Pro Asp Arg Lys Val Leu Leu Ala Gly Ala 20 25 30 Ser Ser Gin Arg Ser Val Ala Arg Met Asp Gly Asp Val Ile lie Gly 35 40 45 Ala Leu Phe Ser Val His His Gin Pro Pro Ala Glu Lys Val Pro Glu 50 55 60 Arg Lys Cys Gly Glu Ile Arg Glu Gin Tyr Gly Ile Gin Arg Val Glu 65 70 75 80 Ala Met Phe His Thr Leu Asp Lys Ile Asn Ala Asp Pro Val Leu Leu 85 90 95 Pro Asn Ile Thr Leu Gly Ser Glu Ile Arg Asp Ser Cys Trp His Ser 100 105 110 Ser Val Ala Leu Glu Gin Ser Ile Glu Phe Ile Arg Asp Ser Leu Ile 115 120 125 Ser Ile Arg Asp Glu Lys Asp Gly Leu Asn Arg Cys Leu Pro Asp Gly 130 135 140 Gin Thr Leu Pro Pro Gly Arg Thr Lys Lys Pro Ile Ala Gly Val Ile 145 150 155 160 Gly Pro Gly Ser Ser Ser Val Ala Ile Gin Val Gin Asn Leu Leu Gin 165 170 175 198 173,552/1

Leu Phe Asp He Pro Gin He Ala Tyr Ser Ala Thr Ser He Asp Leu 180 185 190 Ser Asp Lys Thr Leu Tyr Lys Tyr Phe Leu Arg Val Val Pro Ser Asp 195 200 205 Thr Leu Gin Ala Arg Ala Met Leu Asp He Val Lys Arg Tyr Ash Trp 210 215 220 Thr Tyr Val Ser Ala Val His Thr Glu Gly Asn Tyr Gly Glu Ser Gly 225 230 235 240 Met Asp Ala Phe Lys Glu Leu Ala Ala Gin Glu Gly Leu Cys He Ala 245 250 255 His Ser Asp Lys He Tyr Ser Asn Ala Gly Glu Lys Ser Phe Asp Arg 260 265 270 Leu Leu Arg Lys Leu Arg Glu Arg Leu Pro Lys Ala Arg Val Val Val 275 280 285 Cys Phe Cys Glu Gly Met Thr Val Arg Gly Leu Leu Ser Ala Met Arg 290 295 300 Arg Leu Gly Val Val Gly Glu Phe Ser Leu He Gly Ser Asp Gly Trp 305 310 315 320 Ala Asp Arg Asp Glu Val He Glu Gly Tyr Glu Val Glu Ala Asn Gly 325 330 335 Gly He Thr lie Lys Leu Gin Ser Pro Glu Val Arg Ser Phe Asp Asp 340 345 350 Tyr Phe Leu Lys Leu Arg Leu Asp Thr Asn Thr Arg Asn Pro Trp Phe 355 360 365 Pro Glu Phe Trp Gin His Arg Phe Gin Cys Arg Leu Pro Gly His Leu 370 375 380 Leu Glu Asn Pro Asn Phe Lys Lys Val Cys Thr Gly Asn Glu Ser Leu 385 390 395 400 Glu Glu Asn Tyr Val Gin Asp Ser Lys Met Gly Phe Val He Asn Ala 405 410 415 lie Tyr Ala Met Ala His Gly Leu Gin Asn Met His His Ala Leu Cys 420 425 430 Pro Gly His Val Gly Leu Cys Asp Ala Met Lys Pro He Asp Gly Arg 435 440 445 Lys Leu Leu Asp Phe Leu He Lys Ser Ser Phe Val Gly Val Ser Gly 450 455 460 Glu Glu Val Trp Phe Asp Glu Lys Gly Asp Ala Pro Gly Arg Tyr Asp 465 470 475 480 He Met Asn Leu Gin Tyr Thr Glu Ala Asn Arg Tyr Asp Tyr Val His 485 490 495 Val Gly Thr Trp His Glu Gly Val Leu Asn He Asp Asp Tyr Lys lie 500 505 510 Gin Met Asn Lys Ser Gly Met Val Arg Ser Val Cys Ser Glu Pro Cys 515 520 525 Leu Lys Gly Gin lie Lys Val He Arg Lys Gly Glu Val Ser Cys Cys 530 535 540 Trp He Cys Thr Ala Cys Lys Glu Asn Glu Phe Val Gin Asp Glu Phe 545 550 555 560 Thr Cys Arg Ala Cys Asp Leu Gly Trp Trp Pro Asn Ala Glu Leu Thr 565 570 575 Gly Cys Glu Pro He Pro Val Arg Tyr Leu Glu Leu Arg Glu His Thr 580 585 590 Ser Trp Val Leu Leu Ala Ala Asn Thr Leu Leu Leu Leu Leu Leu Leu 595 600 605 Gly Thr Ala Gly Leu Phe Ala Trp His Leu Asp Thr Pro Val Val Arg 610 615 620 Ser Ala Gly Gly Arg Leu Cys Phe Leu Met Leu Gly Ser Leu Ala Ala 625 630 635 640 Gly Ser Gly Ser Leu Tyr Gly Phe Phe Gly Glu Pro Thr Arg Pro Ala 645 650 655 199 173,552/1

Cys Leu Leu Arg 660 Gin Ala Leu Phe Ala Leu 665 Gly Phe Thr Ile 670 Phe Leu Ser Cys Leu Thr Val Arg Ser Phe Gin Leu Ile Ile Ile Phe Lys Phe 675 680 685 Ser Thr Lys Val Pro Thr Phe Tyr His Ala Trp Val Gin Asn His Gly 690 695 700 Ala Gly Leu Phe Val Met Ile Ser Ser Ala Ala Gin Leu Leu Ile Cys 705 710 715 720 Leu Thr Trp Leu Val Val Trp Thr Pro Leu Pro Ala Arg Glu Tyr Gin 725 730 735 Arg Phe Pro His Leu Val Met Leu Glu Cys Thr Glu Thr Asn Ser Leu 740 745 750 Gly Phe Ile Leu Ala Phe Leu Tyr Asn Gly Leu Leu Ser lie Ser Ala 755 760 765 Phe Ala Cys Ser Tyr Leu Gly Lys Asp Leu Pro Glu Asn Tyr Asn Glu 770 775 780 Ala Lys Cys Val Thr Phe Ser Leu Leu Phe Asn Phe Val Ser Trp Ile 785 790 795 800 Ala Phe Phe Thr Thr Ala Ser Val Tyr Asp Gly Lys Tyr Leu Pro Ala 805 810 815 Ala Asn Met Met Ala Gly Leu Ser Ser Leu Ser Ser Gly Phe Gly Gly 820 825 830 Tyr Phe Leu Pro Lys Cys Tyr Val Ile Leu Cys Arg Pro Asp Leu Asn 835 840 845 Ser Thr Glu His Phe Gin Ala Ser Ile Gin Asp Tyr Thr Arg Arg Cys 850 855 860 Gly Ser Thr 865 &lt;210&gt; 32 &lt;211&gt; 866

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 32

Met Val Arg Leu Leu Leu Ile Phe Phe Pro Met Ile Phe Leu Glu Met 1 5 10 15 Ser Ile Leu Pro Arg Met Pro Asp Arg Lys Val Leu Leu Ala Gly Ala 20 25 30 Ser Ser Gin Arg Ser Val Ala Arg Met Asp Gly Asp Val Ile Ile Gly 35 40 V 45 Ala Leu Phe Ser Val His His Gin Pro Pro Ala Glu Lys Val Pro Glu 50 55 60 Arg Lys Cys Gly Glu Ile Arg Glu Gin Tyr Gly Ile Gin Arg Val Glu 65 70 75 80 Ala Met Phe His Thr Leu Asp Lys Ile Asn Ala Asp Pro Val Leu Leu 85 90 95 Pro Asn Ile Thr Leu Gly Ser Glu Ile Arg Asp Ser Cys Trp His Ser 100 105 110 Ser Val Ala Leu Glu Gin Ser Ile Glu Phe Ile Arg Asp Ser Leu Ile 115 120 125 Ser Ile Arg Asp Glu Lys Asp Gly Leu Asn Arg Cys Leu Pro Asp Gly 130 135 140 Gin Thr Leu Pro Pro Gly Arg Thr Lys Lys Pro Ile Ala Gly Val Ile 145 150 155 160 Gly Pro Gly Ser Ser Ser Val Ala Ile Gin Val Gin Asn Leu Leu Gin 165 170 175 200 173,552/1

Leu Phe Asp He 180 Pro Gin He Ala Tyr 185 Ser Ala Thr Ser He 190 Asp Leu Ser Asp Lys Thr Leu Tyr Lys Tyr Phe Leu Arg Val Val Pro Ser Asp 195 200 205 Thr Leu Gin Ala Arg Ala Met Leu Asp He Val Lys Arg Tyr Asn Trp 210 215 220 Thr Tyr Val Ser Ala Val His Thr Glu Gly Asn Tyr Gly Glu Ser Gly 225 230 235 240 Met Asp Ala Phe Lys Glu Leu Ala Ala Gin Glu Gly Leu Cys lie Ala 245 250 255 His Ser Asp Lys He Tyr Ser Asn Ala Gly Glu Lys Ser Phe Asp Arg 260 265 270 Leu Leu Arg Lys Leu Arg Glu Arg Leu Pro Lys Ala Arg Val Val Val 275 280 285 Cys Phe Cys Glu Gly Met Thr Val Arg Gly Leu Leu Ser Ala Met Arg 290 295 300 Arg Leu Gly Val Val Gly Glu Phe Ser Leu lie Gly Ser Asp Gly Trp 305 310 315 320 Ala Asp Arg Asp Glu Val lie Glu Gly Tyr Glu Val Glu Ala Asn Gly 325 330 335 Gly He Thr lie Lys Leu Gin Ser Pro Glu Val Arg Ser Phe Asp Asp 340 345 350 Tyr Phe Leu Lys Leu Arg Leu Asp Thr Asn Thr Arg Asn Pro Trp Phe 355 360 365 Pro Glu Phe Trp Gin His Arg Phe Gin Cys Arg Leu Pro Gly His Leu 370 375 380 Leu Glu Asn Pro Asn Phe Lys Lys Val Cys Thr Gly Asn Glu Ser Leu 385 390 395 400 Glu Glu Asn Tyr Val Gin Asp Ser Lys Met Gly Phe Val lie Asn Ala 405 410 415 lie Tyr Ala Met Ala His Gly Leu Gin Asn Met His His Ala Leu Cys 420 425 430 Pro Gly His Val Gly Leu Cys Asp Ala Met Lys Pro He Asp Gly Arg 435 440 445 Lys Leu Leu Asp Phe Leu He Lys Ser Ser Phe Val Gly Val Ser Gly 450 455 460 Glu Glu Val Trp Phe Asp Glu Lys Gly Asp Ala Pro Gly Arg Tyr Asp 465 470 475 480 He Met Asn Leu Gin Tyr Thr Glu Ala Asn Arg Tyr Asp Tyr Val His 485 490 495 Val Gly Thr Trp His Glu Gly Val Leu Asn He Asp Asp Tyr Lys He 500 505 510 Gin Met Asn Lys Ser Gly Met Val Arg Ser Val Cys Ser Glu Pro Cys 515 520 525 Leu Lys Gly Gin He Lys Val lie Arg Lys Gly Glu Val Ser Cys Cys 530 535 540 Trp He Cys Thr Ala Cys Lys Glu Asn Glu Phe Val Gin Asp Glu Phe 545 550 555 560 Thr Cys Arg Ala Cys Asp Leu Gly Trp Trp Pro Asn Ala Glu Leu Thr 565 570 575 Gly Cys Glu Pro lie Pro Val Arg Tyr Leu Glu Trp His Glu Ala Pro 580 585 590 Thr He Ala Val Ala Leu Leu Ala Ala Leu Gly Phe Leu Ser Thr Leu 595 600 605 Ala He Leu Val He Phe Trp Arg His Phe Gin Thr Pro He Val Arg 610 615 620 Ser Ala Gly Gly Pro Met Cys Phe Leu Met Leu Thr Leu Leu Leu Val 625 630 635 640 Ala Tyr Met Val Val Pro Val Tyr Val Gly Pro Pro Lys Val Ser Thr 645 650 655 201 173,552/1

Cys Leu Cys Arg 660 Gin Ala Leu Phe Pro 665 Leu Cys Phe Thr He 670 Cys He Ser Cys He Ala Val Arg Ser Phe Gin He Val Cys Ala Phe Lys Met 675 680 685 Ala Ser Arg Phe Pro Arg Ala Tyr Ser Tyr Trp Val Arg Tyr Gin Gly 690 695 700 Pro Tyr Val Ser Met Ala Phe lie Thr Val Leu Lys Met Val He Val 705 710 715 720 Val lie Gly Met Leu Ala Thr Gly Leu Ser Pro Thr Thr Arg Thr Asp 725 730 735 Pro Asp Asp Pro Lys He Thr He Val Ser Cys Asn Pro Asn Tyr Arg 740 745 750 Asn Ser Leu Leu Phe Asn Thr Ser Leu Asp Leu Leu Leu Ser Val Val 755 760 765 Gly Phe Ser Phe Ala Tyr Met Gly Lys Glu Leu Pro Thr Asn Tyr Asn 770 775 780 Glu Ala Lys Phe He Thr Leu Ser Met Thr Phe Tyr Phe Thr Ser Ser 785 790 795 800 Val Ser Leu Cys Thr Phe Met Ser Ala Tyr Ser Gly Val Leu Val Thr 805 810 815 lie Val Asp Leu Leu Val Thr Val Leu Asn Leu Leu Ala He Ser Leu 820 825 830 Gly Tyr Phe Gly Pro Lys Cys Tyr Met lie Leu Phe Tyr Pro Glu Arg 835 840 845 Asn Thr Pro Ala Tyr Phe Asn Ser Met lie Gin Gly Tyr Thr Met Arg 850 855 860 Arg Asp 865 &lt;210&gt; 33 &lt;211&gt; 876

&lt;212&gt; PRT &lt;213&gt; Artificial Sequence &lt;220&gt; &lt;223&gt; Description of Artificial Sequence; note = synthetic construct &lt;400&gt; 33

Met Val Arg Leu Leu Leu lie Phe Phe Pro Met lie Phe Leu Glu Met 1 5 10 15 Ser He Leu Pro Arg Met Pro Asp Arg Lys Val Leu Leu Ala Gly Ala 20 25 30 Ser Ser Gin Arg Ser Val Ala Arg Met Asp Gly Asp Val He lie Gly 35 40 45 Ala Leu Phe Ser Val His His Gin Pro Pro Ala Glu Lys Val Pro Glu 50 55 60 Arg Lys Cys Gly Glu He Arg Glu Gin Tyr Gly He Gin Arg Val Glu 65 70 75 80 Ala Met Phe His Thr Leu Asp Lys He Asn Ala Asp Pro Val Leu Leu 85 90 95 Pro Asn He Thr Leu Gly Ser Glu He Arg Asp Ser Cys Trp His Ser 100 105 110 Ser Val Ala Leu Glu Gin Ser He Glu Phe He Arg Asp Ser Leu He 115 120 125 Ser He Arg Asp Glu Lys Asp Gly Leu Asn Arg Cys Leu Pro Asp Gly 130 135 140 Gin Thr Leu Pro Pro Gly Arg Thr Lys Lys Pro lie Ala Gly Val He 145 150 155 160 Gly Pro Gly Ser Ser Ser Val Ala lie Gin Val Gin Asn Leu Leu Gin 165 170 175 202 173,552/1

Leu Phe Asp lie Pro 180 Gin lie Ala Tyr 185 Ser Ala Thr Ser lie 190 Asp Leu Ser Asp Lys Thr Leu Tyr Lys Tyr Phe Leu Arg Val Val Pro Ser Asp 195 200 205 Thr Leu Gin Ala Arg Ala Met Leu Asp lie Val Lys Arg Tyr Asn Trp 210 215 220 Thr Tyr Val Ser Ala Val His Thr Glu Gly Asn Tyr Gly Glu Ser Gly 225 230 235 240 Met Asp Ala Phe Lys Glu Leu Ala Ala Gin Glu Gly Leu Cys lie Ala 245 250 255 His Ser Asp Lys lie Tyr Ser Asn Ala Gly Glu Lys Ser Phe Asp Arg 260 265 270 Leu Leu Arg Lys Leu Arg Glu Arg Leu Pro Lys Ala Arg Val Val Val 275 280 285 Cys Phe Cys Glu Gly Met Thr Val Arg Gly Leu Leu Ser Ala Met Arg 290 295 300 Arg Leu Gly Val Val Gly Glu Phe Ser Leu lie Gly Ser Asp Gly Trp 305 310 315 320 Ala Asp Arg Asp Glu Val lie Glu Gly Tyr Glu Val Glu Ala Asn Gly 325 330 335 Gly lie Thr lie Lys Leu Gin Ser Pro Glu Val Arg Ser Phe Asp Asp 340 345 350 Tyr Phe Leu Lys Leu Arg Leu Asp Thr Asn Thr Arg Asn Pro Trp Phe 355 360 365 Pro Glu Phe Trp Gin His Arg Phe Gin Cys Arg Leu Pro Gly His Leu 370 375 380 Leu Glu Asn Pro Asn Phe Lys Lys Val Cys Thr Gly Asn Glu Ser Leu 385 390 395 400 Glu Glu Asn Tyr Val Gin Asp Ser Lys Met Gly Phe Val lie Asn Ala 405 410 415 Xie Tyr Ala Met Ala His Gly Leu Gin Asn Met His His Ala Leu Cys 420 425 430 Pro Gly His Val Gly Leu Cys Asp Ala Met Lys Pro lie Asp Gly Arg 435 440 445 Lys Leu Leu Asp Phe Leu lie Lys Ser Ser Phe Val Gly Val Ser Gly 450 455 460 Glu Glu Val Trp Phe Asp Glu Lys Gly Asp Ala Pro Gly Arg Tyr Asp 465 470 475 480 lie Met Asn Leu Gin Tyr Thr Glu Ala Asn Arg Tyr Asp Tyr Val His 485 490 495 Val Gly Thr Trp His Glu Gly Val Leu Asn lie Asp Asp Tyr Lys lie 500 505 510 Gin Met Asn Lys Ser Gly Met Val Arg Ser Val Cys Ser Glu Pro Cys 515 520 525 Leu Lys Gly Gin lie Lys Val lie Arg Lys Gly Glu Val Ser Cys Cys 530 535 540 Trp lie Cys Thr Ala Cys Lys Glu Asn Glu Phe Val Gin Asp Glu Phe 545 550 555 560 Thr Cys Arg Ala Cys Asp Leu Gly Trp Trp Pro Asn Ala Glu Leu Thr 565 570 575 Gly Cys Glu Pro lie Pro Val Arg Tyr Leu Glu Trp Gly Glu Pro Ala 580 585 590 Val Leu Leu Leu Leu Leu Leu Leu Ser Leu Ala Leu Gly Leu Val Leu 595 600 605 Ala Ala Leu Gly Leu Phe Val His His Arg Asp Ser Pro Leu Val Gin 610 615 620 Ala Ser Gly Gly Pro Leu Ala Cys Phe Gly Leu Val Cys Leu Gly Leu 625 630 635 640 Val Cys Leu Ser Val Leu Leu Phe Pro Gly Gin Pro Ser Pro Ala Arg 645 650 655 203 173,552/1

Cys Leu Ala Gin 660 Gin Pro Leu Ser His 665 Leu Pro Leu Thr Gly 670 Cys Leu Ser Thr Leu Phe Leu Gin Ala Ala Glu lie Phe Val Glu Ser Glu Leu 675 680 685 Pro Leu Ser Trp Ala Asp Arg Leu Ser Gly Cys Leu Arg Gly Pro Trp 690 695 700 Ala Trp Leu Val Val Leu Leu Ala Met Leu Val Glu Val Ala Leu Cys 705 710 715 720 Thr Trp Tyr Leu Val Ala Phe Pro Pro Glu Val Val Thr Asp Trp His 725 730 735 Met Leu Pro Thr Glu Ala Leu Val His Cys Arg Thr Arg Ser Trp Val 740 745 750 Ser Phe Gly Leu Ala His Ala Thr Asn Ala Thr Leu Ala Phe Leu Cys 755 760 765 Phe Leu Gly Thr Phe Leu Val Arg Ser Gin Pro Gly Arg Tyr Asn Arg 770 775 780 Ala Arg Gly Leu Thr Phe Ala Met Leu Ala Tyr Phe He Thr Trp Val 785 790 795 800 Ser Phe Val Pro Leu Leu Ala Asn Val Gin Val Val Leu Arg Pro Ala 805 810 815 Val Gin Met Gly Ala Leu Leu Leu Cys Val Leu Gly He Leu Ala Ala 820 82,5 830 Phe His Leu Pro Arg Cys Tyr Leu Leu Met Arg Gin Pro Gly Leu Asn 835 840 845 Thr Pro Glu Phe Phe Leu Gly Gly Gly Pro Gly Asp Ala Gin Gly Gin 850 855 860 Asn Asp Gly Asn Thr Gly Asn Gin Gly Lys His Glu 865 870 875 204 cra&amp;amp;’an ™*a , crnxan rwzn ατα iniab^a pnow pnvn irn πτ qaoa ,ρνιη ηχΰπ naoana ma™ na^maa np’ioz .zruwan rwaa mpnan bmib oxnm □innn bv

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.(moia nannn) cras&amp;amp;'an rwa